Introduction

1. Introduction Double fertilization in flowering plants involves two sperm cells: one fuses with the egg cell to form the zygote, whereas the other one fuses with the central cell to form the endosperm. Plumbago zeylanica is a model system in which it is possible to identify the individual sperm cells prior to fusion. One sperm cell (Svn) is associated with the pollen vegetative nucleus, and the other unassociated sperm cell (Sua) is linked with the Svn. Functionally, the Sua fuses with the egg preferentially over 95% of the time1. The molecular control of this phenomenon is unknown, as gene expression in flowering plant sperm cells is still in its infancy. Work on cells of the male germ linage was impeded by problems obtaining sufficient viable sperm cells and improving their purity during isolation2. To date, we have posted 1,522 ESTs generated from Plumbago sperm cells on GenBank; 893 ESTs of the Sua and 629 ESTs of the Svn, are available at: http://www.genome.ou.edu/plumbago.html. There are also 59 ESTs from rice sperm cells by Fang Chen’s laboratory and 5,000 ESTs of maize sperm cells by Sheila McCormick’s laboratory3 currently available in GenBank. Common features of sperm cell expression are still being determined. A paradigm accepted for decades was that flowering plant sperm cells were simply too small and too dependent to host an independent genetic program. Emerging research in the last decade, however, provided clear evidence of transcription, translation and a unique program3,4,5. A handful of male line-expressed genes have now been characterized, including DNA repair genes6, substitution histones7, and essential sperm specific gene products8. The male gamete-specific expressed gene LGC1, isolated from lily generative cells, is expressed in both generative and sperm cells, its gene product is distributed on the generative cell surface8, and its expression is controlled by a sperm specific promoter9, which has been isolated and expressed in generative cells and sperm cells of lily, tobacco and Arabidopsis. Another sperm-specific promoter has been identified in Arabidopsis and on their website (http://www.pgec.usda.gov/McCormick/McCormick/mclab.html). About 5,000 EST sequences from corn sperm cells are available in GenBank and several sperm cell-specific transcripts have been identified3. Plumbagozeylanica sperm cells undergo an extreme developmental differentiation in organelle content, cellular organization and gene expression during maturation that allows the cells to be discriminated and harvested10 and presumably affects their fate during fertilization. A structural polarization of the developing male germ unit relates the position of sperm cells relative to the pollen vegetative nucleus with structural divergence in which most of the mitochondria and few or no plastids enter the Svn, whereas the Sua contains abundant plastids and few mitochondria1. We are interested in changes in gene expression that relate to their divergence and cell fate during double fertilization. Male germ line-specific ubiquitin expression, which is highly up-regulated in generative cells of lily and in the Svn sperm cell of Plumbago11, has provided a glimpse of such a phenomenon and may relate to differing rates of RNA turnover in the male germ lineage. We are using a genomic approach to identifying genes involved in gamete discrimination that are preferentially up-regulated in just one of the two sperm cells of Plumbago. This is a preliminary and partial characterization of selected clones obtained by combining suppression subtractive hybridization and microarray analysis. Abstract Mature pollen of Plumbago zeylanica contains two sperm cells (Sua and Svn) and one vegetative cell. The two sperm cells have different organelle complements and fuse with different female target cells. The Sua contains more plastids and fuses with the egg cell to form the zygote. The Svn contains more mitochondria and fuses with the central cell to produce the endosperm. To discriminate gene expression differences between the two sperm cells, we constructed representative and subtractive cDNA libraries of these two sperm cell types. The inserts of subtractive cDNA libraries were PCR amplified and spotted onto glass slides for microarray screening. 2304 clones of each subtractive cDNA library were examined using subtracted and unsubtracted cDNA targets. Subtractive hybridization made it easier to identify those genes differentially expressed in these two sperm cell types. Several hundred clones that have differential expression patterns in the two sperm cell types have been identified according to microarray data. Expression patterns of selected clones have been confirmed by real-time RT-PCR. From these candidates, we have obtained several single sperm cell specifically-expressed genes, and their expression specificity has been confirmed by whole mount in situ hybridization in pollen. One sequence is homologous with cytokinin synthase in Arabidopsis and is specifically expressed in Svn. The abundance of cytokinin synthase in the Svn sperm, which fuses with the central cell to form the endosperm, may differentially deliver paternal transcripts contributing to high levels of cytokinin production suspected to stimulate early endosperm activation. Conclusions Our preliminary results reflect abundant sperm-expressed transcripts in mature pollen of Plumbago zeylanica, some with strongly differential expression. These results support that suppression subtractive hybridization and microarray analysis are useful for screening candidates for sperm-type specific expression. Amplification of mRNA through PCR appears to be satisfactory for presence/absence screen-ing; examination of selected clones using RT-PCR and in situ hybridization each agreed with microarray analysis. Sequenced ESTs will be used as the basis for microarray construction in the future with multi-organ and developmental stage sensitive screening used to complete a more comprehensive expressional profiling of sperm-expressed genes. Currently available ESTs indicate that sperm cells have unusually many sequences that are not homologous or are homologous with hypothetical proteins or proteins of unknown function. Although sperm cells have a simple ultrastruct-ural appearance, the male gametic lineage appears to possess a unique, rich and complex system of gene expression, with a seemingly large proportion of sperm unique products. Since the metabolic needs of the male gametic lineage are met by the pollen vegetative cell and tube, presumably a significant proportion of its specific genes govern functions of the sperm cell that are yet poorly understood. Such unique products may include some that are directly involved in governing fusion events of double fertilization, but many others may define other sperm unique characteristics. Zygote/endosperm activation, parent-of-origin epigenetic marking, cell specific recognition, DNA repair, control of RNA expression may all be associated with unique profiles of expression in male gametic cells. Uniquely strongly expressed in Plumbago are genes associated with sperm cell type. Control of such cell-specific activity itself may present unexpected complexity. The diversity of angiosperm reproductive strategies suggests a need for multiple well developed model systems to understand gene expression in sperm cells. The diversity of reproductive strategies suggests a need for instance, for representatives to include monocots & dicots, G1 and G2 fusion systems, bicellular and tricellular pollen. Each model may express different mechanisms controlling their gene expression that cannot be anticipated. In the case of Plumbago, a central question remains as to the control of sperm cell differentiation and its place in flowering plant evolution. Results cDNA libraries construction and EST analysis Initially, we used collections of ≈12,000 sperm cells of each morphotype to construct a size-selected (>400bp) cDNA library for each cell type. A portion of the PCR-amplified cDNA was ligated into lambda TriplEx2 arms and packaged. The titers of the unamplified libraries were 2.1 x 107 and 3.2 x 107 pfu/ml, for Sua and Svn, respectively. The libraries are of high quality: about 90% of the clones had inserts and over 95% of the inserts were at least 500 bp. After sequencing, the ESTs were analyzed and classified into 18 major functional categories of biological process according to Gene Ontology assignments. About 40% of the ESTs of the Sua and Svn could be readily assigned to categories (Figure 1). The two largest categories were “post-translational modification and protein turnover products”, including 20.7% and 29.4% of the Sua and Svn classified products, respectively, and “cell growth/cell division/chromosome partitioning”, which represented 16.8% and 15.0% of the classified products of the Sua and Svn. Among the two sperm morphotypes, the Sua has higher representation of transcription proteins (14.2% vs. 8.9%) and signal transduction products (5.2% vs. 2.2%) than the Svn, whereas the Svn products contain more representatives of “posttranslational modification, protein turnover & chaperone activity” (29.4% compared to 20.7%) and “DNA replication, recombination & repair gene products” (5.0% vs. 1.7%). Figure. 2. Microarray images. Inserts of suppression subtractive hybridization clones were amplified using PCR and spotted on glass slides, forming sperm-expressed probes. Probes in alternating 4-row groups are putative Sua-expressed genes & Svn-expressed genes, respectively. The left slide was hybridized with subtracted targets; the right slide was hybridized with unsubtracted targets. The Sua target was labeled with Cy3 (green), whereas Svn target was labeled with Cy5 (red). A ratio of above 1:4 was used to select differentially expressed clones. Figure. 1. Functional categorization of sequenced ESTs of the Sua and Svn. Four categories conspicuously differ. Not shown are the many sequences with no known homology (43.4% for Sua and 46.9% for Svn) and sequences matching hypothetical and putative proteins (16.5% for Sua and 14.9% for Svn). There were also a small number of retroelements (1.3% for Sua and 1.7% for Svn). Figure 4. Whole mount in situ hybridization of mature pollen of Plumbago zeylanica probed using five different transcripts. Three patterns of sperm cell expression are shown: (1) Up-regulation in both sperm cells. A-C,Clone SuaCon16. D-F, Clone SvnA3F16; (2) Up-regulation in the Sua. G-I, Ubiquitin E2. J-L, Clone SuaCon62; and (3) Up-regulation in the Svn. M-O, Cytokinin synthase. The hybridization signal in sperm cells is clearly visible following digoxigenin labeling with antisense RNA, but not after labeling with the sense probe (P-R). Microscopic imaging methods: brightfield microscopy (left), mixed epifluorescence/brightfield microscopy (center), and epifluorescence microscopy (right) revealing nuclei in pollen. Suppression subtractive hybridization. Suppression subtractive hybridization is a powerful technique for comparing mRNA populations and thereby selecting differentially-expressed genes. Since the quantity of available mRNA is limited for flowering plant sperm cells, we used the Clontech PCR-Select cDNA subtraction method to amplify differentially expressed sequences12. The Sua was used as ‘tester’ and Svn as ‘driver’ for the Sua-differential library and the reverse for the Svn-differential library. Microarray screening was used to select clones that displayed strong preferential binding. Accuracy of screening was later confirmed by more time-consuming northern hybridization and in situ hybridization of a limited number of selected clones. Microarray screening. After suppression subtractive hybridization, PCR products were purified and cloned into pCR2.1-TOPO vector by T/A cloning and transformed into TOP10F’ competent cells. Inserts of the recombinants were amplified using PCR. Most of the recombinants had inserts with sizes ranging from 200 bp to 1.5 kb. A total of 4,608 clones were selected and plotted in alternating groupings of four rows of Sua-differentially expressed sequences followed by four rows of Svn-differentially expressed sequences until all clones were plotted (Figure 2). Microarray screening is based on a competitive hybridization of two complementarily labeled targets: the Sua target labeled with Cy3 and the Svn target labeled with Cy5. Equally hybridized spots should appear yellow, unhybridized spots should appear dark and green and red should represent Sua-differential expression and Svn-differential expression, respectively. To detect low-abundance mRNAs using traditional subtractive hybridization is challenging13,14, so both subtracted and unsubtracted cDNA targets were hybridized with the probes to further contrast differentially expressed sequences15,16. Since insufficient mRNA was available, targets were reverse transcribed and PCR-enhanced prior to fluorescent labeling. Combining suppression subtractive hybridization and microarray analysis techniques facilitates high throughput screening for differentially expressed genes in sperm cells of Plumbago. After analysis by GenePix Pro 3.0, those clones with more than a 4-fold expression difference between the Sua and Svn can be identified numerically using the unsubtracted targets. Typically these clones represent abundant Sua- or Svn-differentially expressed genes. Hybridization with subtracted targets help us to identify low-abundance and rare messages expressed in both the Sua and Svn. Materials and methods Sperm cell isolation Sua and Svn sperm cells were isolated and collected in separate pools using a microinjector, as described in Zhang et al.10. Purified sperm cells were stored in liquid nitrogen until use. About 12,000 sperm cells were collected for each representative cDNA library and subtractive cDNA library.  RNA isolation Total RNA of sperm cells was isolated by using Absolutely RNATM microprep kit (Stratagene) and precipitated by glycogen and ethanol. The RNA pellet was dissolved in 3 µl RNase-free water and immediately used for cDNA synthesis.  cDNA library construction and sequencing cDNA libraries were constructed using the Smart cDNA library construction kit (Clontech) according to the user manual. EST sequencing was carried out at the University of Oklahoma Advanced Center for Genome Technology (ACGT) Lab. Suppression subtractive hybridization Double stranded cDNAs of Sua and Svn were synthesized using Smart PCR cDNA Synthesis kit (Clontech). Subtracted cDNA libraries were constructed using the PCR-Select cDNA Subtraction kit (Clontech). Subtracted cDNAs were purified and cloned into pCR2.1-TOPO vector (Invitrogen).  Microarray experiments Clones of subtracted cDNA libraries were selected randomly and their inserts were PCR amplified. PCR products were purified by ethanol precipitation and dissolved in 50% DMSO. DNA samples were spotted onto CMT-GAPS coated glass slides (Corning) and air dried. Air-dried slides were rehydrated over boiling water and dried again on a 90℃ heating plate. Then slides were crosslinked in Stratagene Stratalinker UV Crosslinker. Slides were prehybridized for 1 hr and hybridized for 16 hr at 42℃ in hybridization chambers. Complementary DNA of Sua and Svn sperm cells were labeled by Cy3 and Cy5 dye (Amersham Phamacia). After washing, the signal was detected by Axon GenePix 4000A microarray scanner.  Real time RT-PCR Because limited materials were used, all the samples were pre-amplified by BD Clontech SMART technology, including microspore, bicellular pollen, root, stem, leaf, mature pollen, sepal, petal, ovary before pollination, ovary after pollination, Sua and Svn. The pre-amplified cDNAs were quantified and 10 ng from each cell/organ were used to carry out real-time PCR. Whole mount in situ hybridization Whole mount in situ hybridization was employed to confirm the differential gene expression between sperm cells in pollen3,7,8. For nonradioactive whole mount in situ hybridization, mature pollen was fixed in 1% glutaraldehyde in 50 mM Pipes buffer (pH7.4) for 2 h at room temperature, rinsed in Pipes buffer and stored in 70% ethanol at 4°C until use. Interesting genes were cloned into pBlueScript SK(+) vector. After the templates are linearized, both sense and antisense riboprobes were labeled with digoxigenin (DIG)-UTP. Hybridization signal were detected using an alkaline phosphatase-conjugated anti-DIG antibody with a DIG nucleic acid detection kit (Roche). Hybridization and signal detection were conducted as described in Singh et al.11. Samples are counter-stained with 4',6'-diamidino-2-phenylindole (DAPI) to visualize nuclei. Acknowledgements We thank Drs. Bruce A. Roe, Tyrrell Conway, Jia Li, Doris M. Kupfer, Hongshing Lai and Mary Beth Langer for their kind help. This research was supported in part by a grant from the USDA NRICGP (#99-35304-8097), the University of Oklahoma and private donations. • References • Russell, SD. 1985. Preferential fertilization in Plumbago: ultrastructural evidence for gamete-level recognition in an angiosperm. Proc Natl Acad Sci USA 82: 6129-6132 • Russell, SD. 1986. A method for the isolation of sperm cells in Plumbago zeylanica. 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Library subtraction of in vitro cDNA libraries to identify differentially expressed genes in scrapie infection. Nucl Acid Res 18: 2789–2792 • Hara E, Kato T, Nakada S, Sekiya S & Oda K. 1991. Subtractive cDNA cloning using oligo(dT)30-latex and PCR: isolation of cDNA clones specific to undifferentiated human embryonal carcinoma cells. Nucleic Acids Res 19: 7097–7104 • Welford SM, Gregg J, Chen E, Garrison D, Sorensen PH, Denny CT, and Nelson SF. 1998.Detection of differentially expressed genes in primary tumor tissues using representational differences analysis coupled to microarray hybridization.Nucleic Acids Res 26: 3059-3065 • Yang GP, Ross DT, Kuang WW, Brown PO, and Weigel RJ. 1999. Combining SSH and cDNA microarrays for rapid identification of differentially expressed genes. Nucleic Acids Res 27: 1517-1523 Real time RT-PCR To demonstrate typical expressional patterns in the sperm cells, several clones were selected for multiple cell/organ real-time RT-PCR. Figure 3 illustrates a number of the observed expression patterns: (1) Gene expressed in all organs or cells to some degree, but with higher levels in sperm cells (Fig. 3A); (2) Expression detected in all organs or cells at different abundance levels (Figs. 3B, C); (3) Genes upregulated exclusively in both sperm cells (Figs. 3D, E); (4) Genes upregulated in Sua (Figs. 3F, G, H); (5) Genes upregulated in Svn (Figs. 3I, J, K, L). Whole mount in situ hybridization Whole mount in situ hybridization was conducted to confirm the differential expression levels between two sperm cells in mature pollen of Plumbago (Fig. 4). These data are consistent with the real-time RT-PCR results. For example, clones SuaCon16 and SvnA3F16 have much higher expression in the sperm cells than in the pollen cytoplasm. Clones SuaCon62 and ubiquitin E2 have much higher expression in the Sua than the Svn. Cytokinin synthase in contrast is expressed essentially exclusively in Svn. A. Histone H3 B. SvnCon46 C. SvnA3N02 D.SvnA3F16 E. SuaCon16 F. SuaCon62 G. E1E12 H. Ubiquitin E2 I. E3H05 J. Cytokinin synthase K. E3E08 L. E4A03 Figure 3. Several genes were selected for further confirmation by real-time RT-PCR. Data were analyzed using ABI Prism 7000 SDS software. In each chart, the samples are (left to right): 1, Microspore; 2, Bicellular pollen; 3, Root; 4, Stem; 5, Leaf; 6, Mature pollen; 7, Sepal; 8, Petal; 9, Ovary before pollination; 10, Ovary after pollination; 11, Sua; 12, Svn. Expression level of Sua was set to 1 and others scaled accordingly.