Double fertilization in flowering plants involves two sperm cells: one fuses with the egg cell to form the zygote, whereas the other fuses with the central cell to form the endosperm.
is a model system in which it is possible to identify the sperm cells. One sperm cell (S vn
) is associated with the pollen vegetative nucleus, and the other unassociated sperm cell (S ua
) is linked with the S vn
. Functionally, the S ua fuses with the egg preferentially over 95% of the time 1 . The molecular control of this phenomenon is unknown, as gene expression in flowering sperm cells is still in its infancy. The first ESTs of genes expressed in sperm cells were released in GenBank in 2000 by Fang
Chen’s laboratory and in 2002 by Sheila McCormick’s laboratory.
Sperm-specific expression of GFP has recently been demonstrated
(http://www.pgec.usda.gov/McCormick/McCormick/mclab.html) in
. The greatest success thus far has been obtained using generative cells, which are the immediate precursors to sperm cells.
Work on cells of the male germ linage was impeded by problems in obtaining sufficient sperm cells to conduct molecular biology and in maintaining their purity during isolation from the pollen 2 . At that time, the accepted paradigm was that the male cells were too small and dependent to have an independent genetic program, but once protein,
RNA and DNA synthesis were demonstrated in sperm 3 and generative cells 4 , work on molecular biology began.
Since then, male gamete expression of DNA repair genes 5 , substitution histones 6 , and essential sperm specific gene products 7 have been demonstrated. The male gametic-specific expressed gene
, isolated from lily generative cells 7 , is expressed in both generative and sperm cells, its gene product is distributed on the generative cell surface, and its expression is controlled by a sperm specific promoter.
We are interested in the control of structural/functional sperm dimorphism, as expressed in
in which the pair of sperm cells undergo a developmental divergence in their organization and gene expression during pollen maturation, which affects their fate during fertilization. The divergence in sperm cells is marked by structural polarization, which is related to the position of the pollen vegetative nucleus and demonstrated most easily by differences in organelle content. The S vn typically contains many mitochondria and few or no plastids, whereas the S ua contains few mitochondria and abundant plastids; the latter usually fusing with the egg cell 1 . We are interested in how the differential fate of these two sperm cells is controlled. Recent research has shown that a male germ line-specific ubiquitin gene is highly up-regulated in both lily generative cells and in the S vn sperm cell of
8 . In this study, we are interested in genes that are exclusively expressed or up-regulated in only one of the two sperm cells of
, especially genes involved in gamete cell recognition. Here we report preliminary results obtained by combining suppression subtractive hybridization and microarray analysis.
Sperm cell isolation
S ua and S vn sperm cells were isolated and collected in separate pools using a microinjector, as described in Zhang et al.
9 Purified sperm cells were stored in liquid nitrogen until use. About 12,000 sperm cells were collected for each cDNA library and subtracted cDNA library.
RNA isolation
Total RNA of sperm cells was isolated by using Absolutely RNA TM 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 cDNA libraries were constructed using the Smart cDNA library construction kit
(Clontech) according to the user manual.
Suppression subtractive hybridization
Double stranded cDNAs of S ua and S vn 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. Subtracted and unsubtracted cDNA of sperm cells were labeled by Cy3 and Cy5 dye (Amersham Phamacia). After washing, the signal was detected by Axon GenePix 4000A microarray scanner.
Virtual Northern hybridization cDNA of sperm cells was synthesized by the Smart PCR cDNA synthesis kit. 0.5 µg dscDNA of S ua and S vn was separated respectively on 1% agarose gel and transferred to nylon membrane. Inserts of interesting clones were labeled by digoxigenin.
Hybridization and signal detection were carried out according to the Roche nonradioactivity detection manual (Roche Applied Science).
RT-PCR
Equal amounts of cDNA of S ua and S vn were used for the RT-PCR reaction. The number of cycles was varied to demonstrate differential expression levels.
1
1
2
1
1 Department of Botany and Microbiology, University of Oklahoma, Norman, OK 73019 USA
2 Plant Molecular & Biotechnology Center, University of Melbourne, Parkville, Victoria 3052, AUSTRALIA
Mature pollen of
contain dimorphic sperm cells of two types: S ua with different female target cells, the S ua fusing with the egg cell and the S vn and S vn
. These cells contain different organelle complements and fuse fusing with the central cell, respectively. To discriminate between genes active in each cell, we constructed subtracted cDNA libraries to each of these two kinds of sperm cells for microarray screening. PCR amplified products were cloned into pCR2.1-TOPO vector. The inserts were PCR amplified and spotted onto glass slides for microarray screening. 2304 clones of each subtracted cDNA library were examined using subtracted and unsubtracted cDNA probes. Subtractive hybridization made it easier to identify those genes expressed differentially in these two kinds of sperm cells. Several hundred clones that have different expression level in a pair of sperm cells have been identified. The expression pattern of seven clones has been verified by virtual northern hybridization and (or) RT-PCR. One of them, clone A7, which is homologous with arabinogalactan protein in
, has a signal peptide sequence and a transmembrane domain. It may play a role in cell recognition.
Because sperm cells are small and embedded in pollen, only limited quantities of sperm cells are available for this research. Therefore, mRNA needs to be reverse transcribed and PCR amplified. To assure that cDNA libraries are representative, PCR reactions need to be controlled carefully. For cDNA library construction, 11,428 S ua sperm cells and 11,073 S vn sperm cells were used. The corresponding PCR cycles were optimized at 24 and 26 cycles, respectively. For subtractive hybridization, 12,200 S ua sperm cells and 12,245 S vn sperm cells were used. The corresponding PCR cycles were optimized at 20 cycles (Fig. 1).

Fig. 1.
Different cycles were used to optimize the PCR reaction of ds-cDNA synthesis of S ua
. M, Molecular standards. 1,
18 cycles. 2, 21 cycles. 3, 24 cycles. 4, 27 cycles. 5, 30 cycles. 6,
33 cycles. 24 cycles were used in this reaction.
Fig. 3 . Microarray. Inserts of suppression subtractive hybridization clones were
PCR amplified and spotted on glass slides. In each block, clones in the top four rows are putative S ua
expressed genes; the next four rows are putative S vn
expressed genes. The left slide was hybridized with subtracted probes; the right slide was hybridized with unsubtracted probes. The
S ua probe was labeled with
Cy3 (green), whereas the
S vn probe was labeled with
Cy5 (red).
Fig. 2 . Inserts of randomly selected clones of subtracted cDNA libraries were PCR amplified and separated on 1% agarose gel.

Subtractive hybridization is a powerful technique for comparing mRNA populations and selecting differentially expressed genes. Traditional subtractive methods are not suitable, as the amount of available mRNA is limited for flowering plant sperm cells. Therefore, the Clontech PCR-Select cDNA subtraction method was used to amplify differentially expressed sequences 10 . S ua
- and
S vn
-specific expressed sequences were obtained by using subtractive hybridization, with one sperm cell source as ‘tester’ and the other as
‘driver’. In cases where different cell sources have strongly overlapping gene expression or where few mRNA species differ, this technique may produce significant numbers of false positives. The S ua and S vn
, as cellular siblings, are thus likely candidates for additional screening. Microarray screening of cell products is an ideal screen prior to time-consuming northern hybridization.
After 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 recombinants were amplified by PCR. Most of the recombinants had inserts whose size ranged from 200 bp to 1.5 kb (Fig. 2). A total of 4,608 clones were selected for the following microarray analysis. Clones corresponding to differentially expressed genes will hybridize only with the tester probe.
But it will be difficult to detect low-abundance differentially expressed mRNAs by traditional subtractive hybridization 11,12 . This problem can be bypassed by using subtracted cDNA probes to hybridize with the subtracted libraries. In this work, subtracted probes were synthesized by using S ua and S vn as tester or driver respectively. Since insufficient mRNA was available, probe was reverse transcribed, PCR-enhanced and labeled using Cy3 or Cy5.
Combining suppression subtractive hybridization and microarray analysis techniques facilitates high throughput screening for differentially expressed genes in sperm cells of
. Most of the clones of subtracted cDNA libraries represent differentially expressed S ua or S vn genes (Fig. 3).
When hybridized with subtracted probes, the signal is stronger than that of unsubtracted probes and some low abundance, differentially expressed sequences can be detected 13,14 . After analysis by GenePix Pro 3.0, those clones with more than a 3-fold expression difference between the S ua
S vn and can be identified easily with unsubtracted probes. Usually these clones represent abundant S ua or S vn specific genes. Hybridization with subtracted probes will help us to identify those low-abundance and rare messages expressed in S ua or S vn
(Table 1).
Table 1.
Number of up-regulated expression clones using different probes
Unsubtracted probes Subtracted probes
Number of up-regulated expression clones in S ua
(Ratio of means>3)
Number of up-regulated expression clones in S vn
(Ratio of means>3)
102
1071
1004
1236
To confirm expression specificity, six
S ua
-specific candidates and one S vn
-specific candidate were selected and digoxigenin labeled. Labeled probes were hybridized with 0.5 µg S ua
S vn cDNA. All of them were confirmed as S ua
- or S vn and
-specifically expressed genes. Four clones (A1, A12, B9, F4) have more than 1 gene family member. These clones were sequenced and compared with the data in
GenBank by BlastX. The results are summarized in Table 2.
Fig. 4.
0.5 µg SMART PCR amplified cDNA of S ua and S vn were separated on 1% agarose gel and transferred to nylon membrane. The blot was hybridized with DIG-labeled probes.
Table 2.
Differentially expressed clones.
Clone
A1
A12
A7
B9
C8
D9
F4
Sequence identity
ATP synthase epsilon subunit [Phyllonoma ruscifolia] homocysteine S-methyltransferase-4 [Zea mays] arabinogalactan-protein (AGP20) [Arabidopsis thaliana]
ATP-dependent Clp protease proteolytic subunit [Nicotiana tabacum] putative DNA-directed RNA polymerase II subunit [Arabidopsis thaliana] hypothetical protein [Arabidopsis thaliana]
Hypothetical ORF [Saccharomyces cerevisiae] mRNA size (Kb)
NA
NA
0.83
NA
0.85
0.95
NA
ACGCCGCCCGTTGACTACTCGTTTCTATCACGCGCTTCCTACCTCCTTCCTCTTTCTTCTTCTTCTTTTTTTTTTCTTTTTTTTCCTGAATGCTTCCTTAGAAATTTCACTGGAGAATCT 121
TCCCCCACTTAATAGTTGAATCGTATATATACGCGTTAGAGAAAGATTTTCTATCGCAAACAATCAAGCCATTCGTACACAGAGTCCTTAATTATTCCACCTCTCTTCCTTTAAATTTCC 241
TCTAATTACTCAGCTTGAGGCAGTTTATTGAGGCACGTCGGCAGGTCGCATCTCTTTCTGAAATACTAATCGTTTGATATAATATTTGACTTCATTCAATTAGGAGATATGGCGAGATCG 361
M A R S
CACGTTCTTCCAATGATTGGGTTCCTGTTCATGGTTATTTTCCGAGTTTGCTCCGGTCAGATCGCTCCTTCTCCGACGGCGGAAGTCCCAGCGTCAAGCGACGGCACTGCAATTGACCAA 481
H V L P M I G F L F M V I F R V C S G Q I A P S P T A E V P A S S D G T A I D Q
GGAATCGCTTACCTGTTGCTTCTGCTCGCTCTCGCCATCACTTACACCTTCCACTGGTAGCAATCTTTTTTCCTATCACTTCCTATTATATCGCTGAAACCGAATGCGGAATAGAAGAAG 601
G I A Y L L L L L A L A I T Y T F H W *
AATATCTATCGATTGTAGAGAGATTTGAGGGTTCGAAATGATTTGTATCTCCCAGCCCAATTCTTTTTAGATTAGAAACCCCCATTTTTTTAATTAATAAATAAATATAAATGTACTGAG 721
ATTTATTTCATTTTTCTTACGAATCTACTGTTACCGTGTAATTTAAATAAGATAGGGCAATTCTTGTGTTTATAAACGACGTTGGTACTTCTCCCATAAAAAAAAAAAAAAAAAAAAAAA 841
AAAAAAA 848
Fig. 5.
Full-length cDNA sequence of Clone A7. It codes a protein with 63 amino acids.
Three clones, A7, A12 and C8, were selected for RT-PCR detection. Different cycles were used. A7 expression can be detected at 18 cycles in the S ua
, but at least 24 cycles are required in the S vn
(Fig. 6). The
A7 gene has a higher expression level in the S ua
. The full-length cDNA of
A7 was isolated by screening S ua cDNA library (Fig. 5). Sequencing showed that A7 codes a protein with 63 amino acids which is homologous to members of an arabinogalactan protein family in
(Fig. 7).
This protein has a signal peptide and a transmembrane domain. Although the precise function of this gene family is unknown, they may play important roles in diverse developmental processes such as differentiation, cell-cell recognition, embryogenesis and programmed cell death 15 .
Fig. 6.
RT-PCR detection of A7, A12, C8 in S ua and S vn
Different cycles are showed on the top of the gel. H3.3 is a
.
highly expressed control.
Fig. 7.
A7, named as PzAGP, is homologous with putative Arabidopsis arabinogalactan proteins AtAGP16 and AtAGP20.
These results clearly indicate the feasibility of using microarray analysis and suppression subtractive hybridization to screen candidates for S ua
- and
S vn
-specific gene expression patterns. Although sufficient mRNA is not available for traditional testing, use of PCR amplification seems to be adequate for presence/absence screening. In the future, we are interested in sperm cell differentiation and gamete cell-cell recognition. We plan to confirm the expression profiles of S ua
- or S vn
-specific expressed genes by
RT-PCR, virtual northern hybridization and
hybridization.
Following this characterization, we plan to search for corresponding genes in
that may be useful for sperm cell labeling and tracing the double fertilization process. Sequence data will be used to identify putative surface-localized proteins that may mediate sperm-egg interactions. This work will serve as the basis of future research on the control of gene expression that establishes the identity of sperm cell types.
We thank Drs. Bruce A. Roe, Tyrrell Conway, Jia Li, Doris M Kupfer and
Mary Beth Langer for their kind help. This research was supported by
USDA NRICGP grant #99-35304-8097, GLC Research Professorship funds from the University of Oklahoma and private donations.
1.
Russell, SD. 1985. Preferential fertilization in Plumbago : ultrastructural evidence for gamete-level recognition in an angiosperm.
Proc Natl Acad Sci USA , 82:6129-6132
2.
Russell, SD. 1986. A method for the isolation of sperm cells in Plumbago zeylanica .
Plant
Physiology , 81: 317-319
3.
Zhang G, Gifford DJ, Cass DD. 1993. RNA and protein synthesis in sperm cells from Zea mays L. pollen.
Sex Plant Reprod 6: 239-243
4.
Blomstedt CK, Knox RB, Singh MB (1996) Generative cells of Lilium longiflorum possess translatable mRNA and functional protein synthesis machinery.
Plant Mol Biol
31:1083-1086
5.
Xu H, Swoboda I, Bhalla PL, Sijbers AM, Zhao C, Ong EK, Hoeijmakers JH, Singh MB
(1998) Plant homologue of human excision repair gene ERCC1 points to conservation of
DNA repair mechanisms. Plant J 13:823-829
6.
Xu H, Swoboda I, Bhalla PL, Singh MB. 1999a. Male gametic cell-specific expression of
H2A and H3 histone genes.
Plant Mol. Biol.
39:607–614
7.
Xu H, Swoboda I, Bhalla P L, and Singh M B. 1999b. Male gametic cell-specific gene expression in flowering plants.
Proc Natl Acad Sci USA , 96: 2554-2558
8.
Singh MB, Xu H, Bhalla P, Zhang Z, Swoboda I, Russell SD. 2002. Developmental expression of polyubiquitin genes and distribution of ubiquitinated proteins in generative and sperm cells.
Sexual Plant Reproduction 14: 325-329
9.
Zhang Z, Xu H, Singh MB, Russell SD. 1998. Isolation and collection of two populations of viable sperm cells from the pollen of P. zeylanica .
Zygote 6: 295-298
10.
Diatchenko L, Lau YFC, Campbell AP, Chenchik A, Moqadam F, Huang B, Lukyanov S,
Lukyanov K, Gurskaya N, Sverdlov ED & Siebert PD. 1996. Suppression subtractive hybridization: A method for generating differentially regulated or tissue-specific cDNA probes and libraries.
Proc. Natl. Acad. Sci. USA 93:6025-6030
11.
Duguid JR & Dinauer MC 1990. Library subtraction of in vitro cDNA libraries to identify differentially expressed genes in scrapie infection.
Nucl Acid Res 18:2789–2792
12.
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
13.
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
14.
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
15.
Gaspar Y., Johnson K., McKenna J., Bacic A, Schultz C. 2001. The complex structures of arabinogalactan-proteins and the journey towards understanding function.
Plant Mol Biol
47: 161-176