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.
Plumbago zeylanica 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.
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.
Abstract
Conclusions
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.
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 screening; examination of selected clones using RTPCR 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 ultrastructural 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).
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 PCRSelect 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 Svndifferential 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.
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) Upregulation 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 (PR). Microscopic imaging methods: brightfield microscopy
(left), mixed epifluorescence/brightfield microscopy (center),
and epifluorescence microscopy (right) revealing nuclei in
pollen.
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
1.
2.
3.
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.
4.
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.
7.
1
2
4.5
0.9
1.8
1.6
1.4
1.2
3.5
0.8
0.7
0.6
0.4
0.3
0.2
0
A. Histone H3
3
0.8
0.6
0.2
0
0.4
1.5
1
0.2
0.5
B. SvnCon46
9.
0
0
0.5
0.4
0.3
0.2
0.1
0
0.4
8.
0.6
2
1
0.9
0.8
0.7
0.6
1
1
0.8
2.5
0.2
0
0.1
6.
4
1
0.8
0.6
0.4
0.5
5.
C. SvnA3N02
D.SvnA3F16
1
1
0.9
0.9
0.8
0.8
0.7
0.7
0.6
0.6
0.5
0.5
0.4
0.4
0.3
0.3
0.2
0.2
0.1
0.1
0
0
10.
11.
12.
E. SuaCon16
F. SuaCon62
30
10
G. E1E12
10
H. Ubiquitin E2
12
9
25
8
7
8
8
6
5
15
4
10
3
2
5
13.
10
20
6
6
4
4
2
14.
2
1
0
0
I. E3H05
J. Cytokinin synthase
0
0
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.
15.
16.
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. Plant Physiol 81: 317-319
Engel ML, Chaboud A, Dumas C and McCormick S. 2003. Sperm cells of Zea mays have a complex
complement of mRNAs. Plant J 34: 697-707
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
Zhang G, Gifford DJ, Cass DD. 1993. RNA and protein-synthesis in sperm cells isolated from Zea mays L.
pollen. Sex Plant Reprod 6: 239-243
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
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
Xu H, Swoboda I, Bhalla PL, and Singh MB. 1999b. Male gametic cell-specific gene expression in flowering
plants. Proc Natl Acad Sci USA 96: 2554-2558
Singh M, Bhalla PL, Xu H, Singh MB. 2003. Isolation and characterization of a flowering plant male gametic
cell-specific promoter. FEBS 542: 47-52
Zhang Z, Xu H, Singh MB, Russell SD. 1998. Isolation and collection of two populations of viable sperm cells
from the pollen of Plumbago zeylanica. Zygote 6: 295-298
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. Sex Plant Reprod 14: 325-329
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
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
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