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First published online 1 November 2006
doi: 10.1242/dev.02683
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1 Howard Hughes Medical Institute, Department of Molecular Genetics and Cell
Biology, The University of Chicago, Chicago, Il 60637, USA.
2 Medical Scientist Training Program, The University of Chicago, Il 60637,
USA.
3 Department of Molecular Biology, Cell Biology and Biochemistry, Brown
University, Providence, RI 02912, USA.
* Author for correspondence (e-mail: Mark_Johnson_1{at}Brown.edu)
Accepted 4 October 2006
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SUMMARY |
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 TOP SUMMARY INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Key words: Arabidopsis, Pollen tube guidance, Double fertilization, Sperm, Gamete interactions
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INTRODUCTION |
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TOPSUMMARYINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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).
The pollen tube interacts with many female sporophytic cells on its journey
to the FG (Johnson and Preuss,
2002). The stigma surface binds pollen from a select set of
species and subsequently perceives pollen signals that trigger the controlled
hydration of the pollen grain, enabling tube germination
(Swanson et al., 2004).
Guidance cues provided by the female sporophytic tissues determine the initial
polarity of pollen tube extension
(Kandasamy et al., 1994;
Kim et al., 2003;
Lord, 2003;
Park and Lord, 2003;
Wolters-Arts et al., 1998),
directing its growth through the extracellular matrix of the style and
transmitting tract (Lord,
2003; Wu et al.,
2000). After a pollen tube exits the transmitting tract, it
emerges onto the septum and grows towards an ovule, navigating up the
funiculus and into the micropyle, which provides access to a FG. The FG is a
seven-celled haploid structure comprising an egg and two synergid cells at the
micropylar pole, a large central cell, and three antipodal cells
(Yadegari and Drews, 2004).
Pollen tube growth arrests within a synergid and the pollen tube tip bursts,
releasing two sperm (Russell,
1992). This process, called pollen tube reception, is accompanied
by the degeneration of the receptive synergid and is rapidly followed by the
fusion of one sperm with the egg and one sperm with the central cell to
produce the zygote and endosperm, respectively (reviewed in
Faure and Dumas, 2001;
Weterings and Russell,
2004).
Pollen tube guidance in the ovary is controlled by factors expressed by
diploid floral cells and by haploid FG cells
(Christensen et al., 2002;
Hülskamp et al., 1995;
Pagnussat et al., 2005;
Palanivelu et al., 2003;
Ray et al., 1997;
Shimizu and Okada, 2000).
Genetic experiments in Arabidopsis show that pollen tubes bypass
incompletely formed ovules or those that lack a FG
(Hülskamp et al., 1995;
Ray et al., 1997). When ovules
carry maa1 or maa3, mutations that delay FG development
(Shimizu and Okada, 2000),
pollen tubes grow up the ovule funiculus but fail to enter the micropyle,
suggesting that the FG produces distinct signals for funicular and micropylar
guidance. Laser-ablation studies combined with an elegant in vitro pollen tube
guidance assay using Torenia fournieri, showed that the synergids
produce a short-range micropylar attractant
(Higashiyama et al., 2001). A
candidate FG-derived pollen tube attractant has recently been identified;
Zea mays egg apparatus1 (ZmEA1) is expressed exclusively in
egg and synergid cells, and encodes a 94 amino acid hydrophobic protein that
is required for efficient micropylar guidance in a maize in vitro guidance
system (Marton et al.,
2005).
FG signals may act in concert with sporophytic pollen tube attractants
produced by ovule integuments or by the funiculus. 
-amino butyric acid
(GABA) may be one such signal; Arabidopsis pop2 mutants disrupt the
GABA gradient near the micropyle, resulting in random pollen tube growth
(Palanivelu et al., 2003).
Additional signaling events take place after the pollen tube enters the
micropyle. When pollen tubes enter ovules bearing feronia or
sirene mutant FGs, the synergid degenerates, but the pollen tube does
not stop growing and does not burst, suggesting that the FG produces a signal
that controls pollen tube reception (Huck
et al., 2003; Rotman et al.,
2003). These results represent tremendous progress in
understanding the female signals that guide the pollen tube to the ovule;
however, there is little information on how the pollen tube perceives these
signals and transmits them into changes in the direction of its growth.
To identify pollen-expressed genes that are important for pollen tube
growth and guidance, we isolated a series of MG mutants by screening for
distorted inheritance (Johnson et al.,
2004). Mutants were induced by random genomic insertions of a
T-DNA carrying an herbicide-resistance gene (Basta resistance,
BastaR), as well as a cell-autonomous histochemical marker
(ß-glucoronidase, GUS) under the control of the post-meiotic
pollen-specific promoter LAT52 (Twell et
al., 1989). LAT52 is active during pollen tube growth, marking
mutant pollen tubes from their initial interactions with stigmatic papillae
cells to their arrival at an FG. Mendelian inheritance predicts that plants
with one copy of the T-DNA insertion will produce 75% BastaR
offspring when self-fertilized. By contrast, self-fertilization of
hap2/HAP2 plants yielded 
50% BastaR progeny and no
hap2/hap2 homozygotes were obtained
(Johnson et al., 2004). This
transmission defect was male-specific; reciprocal crosses of
hap2/HAP2 with wild type generated BastaR progeny at 0.7%
or 47.0% when hap2/HAP2 was used as the male or female, respectively
(Johnson et al., 2004).
Initial phenotypic characterization of hap2 pollen tube growth
suggested that hap2 pollen tubes do not follow the wild-type path,
growing chaotically within the ovary. We identified the hap2 T-DNA
insertion site within a hypothetical gene of unknown function (At4g11720)
(Johnson et al., 2004).
Recently, a generative cell-specific protein (GCS1) from lily was characterized, and a T-DNA insertion into its Arabidopsis homolog, At4g11720 (HAP2), indicated a role in fertilization. GCS1 is localized to sperm cells, and we confirm here that a HAP2-fusion protein is sperm-expressed. Importantly, with the LAT52-GUS insertion into HAP2, we were able to look at phenotypes prior to gamete fusion, showing that HAP2 is also required for pollen tube guidance. hap2 pollen tube length is not affected. However, hap2 pollen tubes are half as likely as wild type to target ovules; after leaving the septum, hap2 tubes meander over ovule surfaces where wild-type pollen tubes do not typically grow. In the cases where hap2 pollen tubes reach ovules, hap2 completely blocks fertilization. This study thus provides genetic evidence for the active participation of sperm cells in their delivery to female gametes.
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MATERIALS AND METHODS |
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TOPSUMMARYINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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) and
control lines in the Col-0 background were grown at 21°C in 24-hour 100
µE fluorescent lighting or in ambient greenhouse conditions. Kanamycin
resistance (KanR; hap2-2) was determined on MS salts with
25 µg/ml kanamycin, and BastaR (hap2-1) was determined
as by Johnson et al. (Johnson et al.,
2004). Plant transformation was performed as described
(Clough and Bent, 1998),
selecting for growth on 50 µg/ml kanamycin or on soil with 1:2000 Liberty
herbicide (AgrEvo, Pikeville NC). Limiting pollinations were performed with
male sterile (ms1) pistils and hap2/HAP2 anthers; crosses
producing 25 or fewer seeds were analyzed.
The hap2-1 T-DNA-gene junction was confirmed with the PCR primers
LB3 (McElver et al., 2001),
HAP2 upstream (5'-GGCCTCACTCGTTCTCAATTGGAG-3') and
HAP2 downstream (5'-GTGAGAGTCGCTGTGGTCACGTTC-3'). The
hap2-2 T-DNA-gene junction was confirmed with the PCR primers LBa1
(Alonso et al., 2003) and
HAP2seqTR3 (5'-CAATCAAACTGCGCAGAAGGAAGC-3'). Full-length
HAP2 cDNA (GenBank DQ022375) was amplified from 200 ng total pollen
RNA using primers F, 5'-AAACAATTTTCAATTCGCGTCTCCG-3' and R,
5'-AACTCGGATATATTTTTGC-3'. For complementation, a genomic
HAP2 fragment was PCR amplified with F,
5'-GGCCCTGCAGGTTCTGATCCTAACAACAACGGCGGC-3', and R,
5'-CCGCCTGCAGGATATCTTCGAGAGAATCACCAGTCGCC-3', inserted into the
SbfI site of pCAMBIA2300 (GenBank AF234315), sequenced, introduced
into Agrobacterium (strain GV3101), and transformed into
hap2-1/HAP2 plants, selecting KanR.
Protein sequence analysis
The Arabidopsis HAP2 coding sequence guided annotation of the
Poplar ortholog from genomic sequence (Poptr1:63588;
http://genome.jgi-psf.org).
Other sequences were obtained from GenBank (accession number): Lilium
longiflorum (AB206810), Oryza sativa (AK072871),
Chlamydomonas reinhardtii (AB206813), Cyanidioschyzon
merolae (AP006493), Plasmodium falciparum (AAN35337),
Physarum polycephalum (AB206812), Leishmania major
(CP000081) (Mori et al.,
2006). Predicted proteins were aligned using ClustalW (MegAlign,
DNAstar, Madison, WI), and structure and subcellular localization was
predicted with PSORT (Nakai and Kanehisa,
1992), TargetP (Emanuelsson et
al., 2000), Genoplante Predator
(http://www.genoplante.com/content.php?idcontent=bioinfotools&lg=en),
Prosite
(http://us.expasy.org)
and TMHMM
(http://www.cbs.dtu.dk).
Phenotypic analysis
Pollen tube growth in pistils was analyzed as previously described
(Johnson et al., 2004). For in
vitro analyses, pollen from stage-14 flowers
(Smyth et al., 1990) was
germinated for 3 hours in an inverted drop of medium
(Hicks et al., 2004). Pollen
tubes or grains were transferred to polylysine-coated slides and stained with
DAPI (Park et al., 1998),
Aniline Blue (0.01% in 50 mM KPO4, 50% glycerol), FM 4-64 (3.4 µM in
germination medium) or X-Gluc (for GUS activity)
(Johnson et al., 2004). All
images were captured on a Zeiss Axioskop (Carl Zeiss, Germany); pollen tube
lengths were measured using ImageJ
(http://rsb.info.nih.gov/ij/).
Fertilization and embryo development was analyzed as previously described
(Yadegari et al., 1994);
indicated samples were first stained for GUS activity
(Johnson et al., 2004).
HAP2 expression
Northern blotting was performed on 20 µg of total pollen RNA probed with
HAP2 cDNA. RT-PCR was performed on 1 µg total pollen RNA using the
HAP2 primers F, 5'-TTAATGGCTTGTATACTCGCCGG-3' and R,
5'-ACGAAGGCAATGCGCGGTATTTGCC-3', and EF-1
controls
F, 5'-GCCCCTTCGTCTCCCACTTC-3' and R,
5'-CACTTCGCACCCTTCTTGACG-3'; products were analyzed after 25, 30,
35 and 40 PCR cycles. The HAP2promoter:YFP fusion contained 983 bp
upstream of the HAP2 start codon, amplified using HAP2SacIF,
5'-GGCGAGCTCAATTTCCCTGATACAATCCCGAGGC-3' and HAP2BamHIR,
5'-GGCGGATCCTTTCTCTCTCACGGAGACGCG-3'; HAP2protein:YFP
fusion contained the HAP2 promoter and all exons and introns, and was
amplified using primers HAP2SacIF and HAP2NcoR,
5'-GGCCCATGGTACTCTCACGTAGTCTTTGTTTCCTC-3'. Amplified products were
digested with SacI and BamHI, or SacI and
NcoI, respectively, and introduced upstream of eYFP (Clonetech)
followed by the CaMV polyadenylation sequence, and incorporated into the
binary vector, pGREENII02229 (Hellens et
al., 2000).
Transgenic plants were analyzed using a Zeiss Axioplan 200 equipped with a
Photometrics cooled CCD camera (CoolSNAP fxHQ, Roper Scientific, Tuscon, AZ),
or a confocal fluorescence microscope (SP2 A OBS, Leica Microsystems AG,
Wetzlar, Germany) and analyzed with Openlab software (Improvision, Lexington,
MA). For analysis of YFP fluorescence during pollen development, uninucleate,
bicellular and tricellular pollen grains were released from anthers and
stained with DAPI (Park et al.,
1998). They were then analyzed using a Leica DMIRE2 Confocal
microscope with a 63 x water objective using Leica software. DAPI was
excited with a 405 nm laser and detected at 410-581 nm, whereas the YFP was
excited with a 514 nm laser and detected at 517-597 nm. Pollen tubes were
grown for up to 6 hours in liquid media on an inverted microscope slide
(Hicks et al., 2004).
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RESULTS |
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TOPSUMMARYINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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To define the stage at which hap2 pollen tubes fail, we monitored
the path of hap2-1 pollen tubes in the ovary and determined the
frequency with which hap2-1 pollen tubes successfully enter a
micropyle and burst. At 14 hours after hand-pollinating ms1 pistils
with control anthers heterozygous for the LAT52:GUS reporter, GUS activity was
observed in the synergid cells of approximately half of the ovules, as
expected (116/234, 50%, Fig.
1B,C). By contrast, when hap2-1/HAP2 anthers were used, a
smaller proportion of GUS+ pollen tubes entered a micropyle and
burst (115/505, 23%; Fig.
1B,D). Furthermore, hap2-1 pollen tubes were observed
growing on portions of the ovule where wild-type pollen tubes do not grow
(Fig. 1E) and also stalled on
the funiculus (Fig. 1F). These
data indicate that hap2 pollen tubes have a diminished capacity to
enter the micropyle and burst within synergids (50% reduction from wild
type) because of defects in funicular and/or micropylar guidance.
The hap2 ovule-targeting defect was not suppressed when
competition from wild-type pollen tubes was decreased. We pollinated wild-type
pistils with single hap2-1/HAP2 tetrads and, after 14 hours, fixed
and stained the pistils with Aniline Blue to follow the route of
hap2-1 and HAP2 pollen tubes. With a control tetrad donor,
100% of the pollen tubes that germinated were able to target and enter an
ovule (n=35). By contrast, 69/89 (78%) of the pollen tubes from
single hap2-1/HAP2 tetrads targeted an ovule, a significant
difference from wild type (P<0.05, 
2). These
experiments indicate that only half of hap2 pollen tubes reached
their target - the same result as that obtained with excess pollinations.
Thus, the observed hap2 pollen tube-guidance defect is not caused by
an inability to compete with wild-type pollen tubes for access to ovules, but
rather reflects an inherent pollen tube-guidance defect.
hap2 completely blocks fertilization
When hap2-1 or hap2-2 pollen were used to pollinate
wild-type pistils, no mutant progeny were recovered (hap2-1, 363
F1 tested; hap2-2, 348 F1 tested), indicating
that hap2 completely blocks transmission of the mutant allele through
the male. Because hap2 disrupts pollen tube guidance, we addressed
whether competition for available ovules from HAP2 pollen tubes masks
rare hap2 fertilization events by performing limiting pollinations of
wild-type pistils. We obtained 87 F1 seeds from ten crosses: none
inherited the hap2-1 allele. This absolute block in transmission
through pollen cannot be explained by an approximately 50% reduction in the
ability of hap2 pollen tubes to target ovules. Therefore,
hap2 must also disrupt a step in reproduction that occurs after the
contents of the pollen tube are released into the synergid cell of the FG.
We examined the development of seeds in self-fertilized hap2/HAP2 flowers and found that, hap2-1/HAP2 plants averaged 37±7 seeds (742 seeds in total, 20 siliques), whereas HAP2/HAP2 plants average 56±8 seeds (564 seeds in total, ten siliques). hap2-1/HAP2 siliques had gaps (Fig. 2A,B) where ovules failed to develop into seeds, suggesting that the FGs in ovules targeted by hap2 pollen tubes do not get fertilized.
To test this hypothesis, we pollinated wild-type pistils with hap2-1/HAP2 pollen, allowed 48 hours for fertilization, early embryo and endosperm development, and then stained them for GUS activity to identify ovules that had been targeted by hap2-1 pollen tubes. Ovules targeted by HAP2 pollen tubes (no GUS activity) contained early globular embryos and obvious proliferation of endosperm nuclei (122/132 ovules analyzed were fertilized; Fig. 2D). By contrast, ovules targeted by hap2-1 pollen tubes (GUS activity in the synergid cell indicating that the pollen tube cytoplasm and sperm had been deposited) showed no indication of embryo or endosperm development (0/26 ovules analyzed were fertilized; Fig. 2F). Instead, we consistently observed only the unfertilized central cell nucleus in FGs targeted by hap2-1 pollen tubes. We analyzed 112 unstained ovules in wild-type pistils pollinated with hap2-1/HAP2 pollen and found that 18% contained only one central cell nucleus (Fig. 2G), and that the remaining 82% contained an embryo and several endosperm nuclei (Fig. 2E); these values are consistent with the rate of hap2-1 ovule targeting and, when combined with the data from GUS-stained ovules, suggest that when hap2 sperm are released into the FG, they fail to fertilize the egg and central cell.
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). In ovules
targeted by hap2 pollen tubes, the central cell nucleus is the only
FG nucleus that remains 48 hours after pollination. We hypothesized that, in
the absence of fertilization, the egg nucleus degenerates and that the second
synergid degenerates shortly after the pollen tube bursts. Analysis of embryo
and endosperm development at an earlier time point (24 hours after
pollination) showed that both the egg and central cell remain in ovules
targeted by hap2 pollen tubes
(Fig. 2H), whereas an embryo
and endosperm nuclei are clearly present in ovules targeted by HAP2
pollen tubes (Fig. 2I). These
results confirm that hap2 sperm are incapable of fertilizing the egg
or central cell and suggest that, in the absence of fertilization, the egg
cell degenerates following pollen tube reception. Similarly, Mori et al. found
that gcs1 sperm fail to fertilize and that they persist in the
degenerating synergid 16 hours after pollination, whereas wild-type sperm do
not (Mori et al., 2006).
Presumably, wild-type sperm were not observed because they are rapidly
transported to the egg and central cell where they bind these target cells and
undergo plasmogamy.
hap2 sperm develop normally and migrate to the pollen tube tip
To determine whether the hap2 fertilization defect is due to a
defect in sperm development or in migration of sperm within the pollen tube,
we analyzed hap2 pollen grains and tubes throughout their
development. DAPI-staining revealed morphologically normal sperm and
vegetative nuclei (n>500, Fig.
3A-D). Staining hap2/HAP2 pollen tubes either with
Aniline Blue, which binds callose (ß-1-3-glucan) in the pollen tube
walls, or FM 4-64, which becomes incorporated into the membrane architecture
of growing tubes, showed that hap2 pollen tubes were
indistinguishable from wild type (data not shown). In newly germinated tubes,
the vegetative nucleus consistently exited the pollen grain before the two
sperm (hap2/HAP2, n=24; control, n=19), and, as tubes
elongated, the sperm and vegetative nuclei were always in the subapical region
of the tube (hap2/HAP2, n=138; control, n=117). Within 1
hour after pollination, hap2 vegetative nuclei emerged from the
pollen grain ahead of the two sperm (n=11); this normal male
germ-unit organization was maintained as the tubes migrated through the
transmitting tract (n=20). These results indicate that defects of
hap2 pollen tube guidance and fertilization are not the result of
aberrant pollen tube structure or sperm transport within the pollen tube.
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). No
cDNAs or ESTs corresponding to this gene were present in public databases, so
we used 5' and 3' RACE to generate a full-length cDNA and to
annotate At4g11720, determining that the gene comprises 17 exons instead of
the 14 predicted by the initial annotation of the Arabidopsis genome
(GenBank DQ022676 and DQ022375; Fig.
4A). With PCR, we confirmed both hap2-1 T-DNA-genome
junctions, as well as a 10 bp genomic deletion at the insertion site (exon
15). A second allele, hap2-2, with a T-DNA insertion in exon 9
(Fig. 4A) was identified from
the SIGNAL collection (Alonso et al.,
2003); this allele also caused distorted T-DNA segregation (46.2%
KanR progeny from self-fertilization, n=600) and
completely blocked transmission through pollen (0 KanR progeny when
crossed with wild type, n=348). The location of the gcs1
allele is also indicated (Fig.
4A) (Mori et al.,
2006). To confirm that At4g11720 is indeed HAP2, we complemented the hap2 defect by transforming plants heterozygous for hap2-1 with a T-DNA carrying NPTII (conferring KanR) and a wild-type HAP2 transgene (HAP2tr, a genomic fragment from 983 bp upstream to 277 bp downstream of the open reading frame; Fig. 4A). The progeny of primary hap2/HAP2 transformants were collected and BastaR and KanR were analyzed in ten transgenic families with a single-locus insertion of HAP2tr. The average rate of BastaR among these families was 68.6% (n=2362) and of KanR was 83.1% (n=2525). Complementation of both ovule-targeting and fertilization defects is expected to yield T2 progeny that segregate 67% (8/12) BastaR and 83% (10/12) KanR; failure would lead to approximately 50% BastaR, as observed in the progeny of self-fertilizing hap2-1/HAP2 plants. These results indicate that wild-type At4g11720 rescues the hap2-1 pollen tube-guidance and fertilization defects. Self-fertilization of hap2/HAP2;HAP2tr plants yielded hap2/hap2 homozygous progeny (all four members of tetrad are GUS+), the progeny of which were 100% BastaR; homozygous progeny were never observed when hap2/HAP2 is self-fertilized.
|
). Pair-wise
comparison of amino acid identity between the Arabidopsis HAP2 and
HAP2 from other flowering plants showed that the N-terminal region is
conserved (mean identity 64%), whereas the C-terminal region is more divergent
(mean identity 35%). An alignment of the C-terminal histidine-rich regions
showed that the presence of histidine is conserved among these proteins, but
that amino acid sequence is not (Fig.
4C). Interestingly, these histidine-rich domains are present in
HAP2 from angiosperms, but not from other organisms
(Fig. 4D).
HAP2 is only expressed in sperm
RT-PCR and northern blot analysis showed that HAP2 mRNA is only
detected in tissue samples that contain mature pollen
(Fig. 5A,B). This specific
expression pattern is confirmed by hundreds of publicly available microarray
experiments showing that HAP2 mRNA only accumulates in pollen
(https://www.genevestigator.ethz.ch/).
To determine the precise location of HAP2 expression, we fused a DNA
fragment corresponding to 983 bp upstream of the HAP2 start codon
(Fig. 4A) to the yellow
fluorescent protein (YFP) coding sequence (HAP2promoter:YFP fusion
protein) and generated transgenic plants expressing this construct.
Accumulation of YFP was only observed in the two sperm cells contained within
the cytoplasm of mature pollen grains (Fig.
5C,D); YFP was not observed in uninucleate microspores or in
bicellular pollen (Fig. 5D),
nor was it observed in other floral or vegetative cells (data not shown).
Protein localization algorithms predict that HAP2 is localized to the plasma membrane and/or the endoplasmic reticulum (ER). To determine where HAP2 is localized within sperm, we fused YFP to the penultimate codon of HAP2 exon 17 (Fig. 4A, HAP2protein:YFP fusion protein) and generated transgenic plants expressing this construct. Three independent transgenic lines were crossed with hap2-1/HAP2 plants and homozygous hap2-1 lines were obtained in the F2 generation of each cross, indicating that HAP2protein:YFP complemented hap2 pollen tube-guidance and fertilization defects, and suggesting that HAP2protein:YFP forms a functional and properly localized protein. We detected the fusion protein only in the sperm cells (Fig. 6A-D). Unlike the transcriptional fusion, which produced YFP distributed throughout the elongated, spindle-shaped sperm cytoplasm (Fig. 5C), the protein fusion was excluded from the nucleus and was predominantly perinuclear, forming a ring of fluorescence around sperm nuclei (Fig. 6B). However, fluorescence was not limited to the perinuclear region and in many sperm cells, fluorescence extended to the sperm plasma membrane (Fig. S1 in the supplementary material). These results, in combination with protein-localization predictions, suggest that HAP2 is predominantly localized to sperm ER membranes and that HAP2 may also reside in other endomembranes, including the plasma membrane.
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DISCUSSION |
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TOPSUMMARYINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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), we have shown that hap2-1 pollen tubes grow the
entire length of the pistil, yet grow aberrantly on ovule surfaces and often
fail to enter the micropyle, resulting in a twofold reduction in ovule
targeting. In vitro, hap2-1 pollen tubes grow slightly longer and, in
the pistil, hap2-1 pollen tubes grow slightly faster than wild-type
pollen tubes. This mild enhancement of pollen tube extension could reflect the
lack of a tube-growth behavior that is required for optimum guidance.
Interestingly, the ability of hap2 pollen tubes to target ovules does
not improve when pistils are underpollinated, indicating that the
hap2 pollen-tube-guidance defect is not a consequence of an inability
of hap2 pollen tubes to compete with HAP2 pollen tubes for a
limited number of ovules. Thus, hap2 specifically disrupts pollen
tube guidance without diminishing pollen tube growth, making this mutant a
unique resource for understanding how pollen tubes perceive and respond to
guidance cues.
|
). gcs1 pollen tubes are not tagged and analysis of
pollen tube guidance by staining mutant and wild-type tubes with Aniline Blue,
particularly when many pollen tubes are present, is not sufficiently sensitive
to detect hap2 guidance differences. Nonetheless, consistent with our
observations, pollinations of wild-type pistils with gcs1/GCS1 pollen
always yielded a greater number of fully formed (targeted by GCS1
pollen) than aborted seeds (targeted by gcs1 pollen). These data
suggest that the gcs1 allele may also disrupt pollen tube
guidance.
HAP2 is a sperm-specific protein
HAP2 mRNA only accumulates in mature pollen
(Fig. 5A,B), HAP2
promoter activity is only detected in mature sperm
(Fig. 5C,D), and HAP2 protein
is only detected in the sperm during pollen development and tube growth
(Fig. 6). In lily, expression
of the HAP2 ortholog GCS1 is first apparent in the
generative cell and persists after the generative cell divides during pollen
tube growth to produce two sperm (Mori et
al., 2006). This earlier onset of HAP2 expression
probably reflects a difference in pollen development between these species. In
Arabidopsis (which has tricellular pollen grains), the generative
cell divides to produce two sperm before anthesis, whereas, in lily and other
species with bicellular pollen grains, the generative cell does not divide
until after the pollen tube has germinated. These expression studies, combined
with the hap2-mutant phenotype and the finding that HAP2 is
conserved among a diverse set of angiosperms
(Fig. 4D)
(Mori et al., 2006), support
the hypothesis that HAP2 is a sperm-specific gene that is universally
essential for double fertilization.
HAP2 is predicted to encode a 705 amino acid protein that shares
no similarity with proteins of known function. However, it has three features
that offer clues to its biochemical function; HAP2 has an N-terminal cleavable
signal sequence, a single transmembrane domain and a C-terminal histidine-rich
domain (Fig. 4B). Multiple
algorithms predict that HAP2 is an integral ER or plasma-membrane protein and
that the C-terminus is cytoplasmic (type 1a membrane protein). Lily GCS1 (HAP2
ortholog) was shown to be associated with membranes by cellular fractionation
studies (Mori et al., 2006).
The HAP2protein:YFP fusion protein we generated complements
hap2-1, suggesting that it encodes a fully functional protein and
that its localization pattern is that of the endogenous protein.
HAP2protein:YFP is predominantly localized in a perinuclear ring
(Fig. 6B) (consistent with ER
localization), with extensions of HAP2protein:YFP to the plasma
membrane (Fig. S1 in the supplementary material). These results are consistent
with those recently obtained by immunofluorescence in both lily and
Arabidopsis (Mori et al.,
2006), and suggest that HAP2 is associated with membranes of the
perinuclear ER and, perhaps, with secretory vesicles bound for the plasma
membrane. Further studies using higher-resolution techniques will be required
to determine the precise HAP2 localization pattern within sperm cells.
Potential roles for sperm-expressed HAP2 in pollen tube guidance
We have shown that HAP2 is required for pollen tube guidance and that it is
sperm-specific. These results point to a previously unrecognized role for
sperm in directing the growth of the pollen tube. The ability of a sperm
protein to alter the efficiency of pollen tube guidance may suggest a
checkpoint or sperm quality-control mechanism, such that pollen tubes carrying
defective sperm could not efficiently target ovules.
HAP2 may be directly involved in transducing a pollen tube-guidance cue
(Johnson and Lord, 2006). The
sperm are associated with the pollen tube cytoskeleton and migrate near the
tip of the pollen tube as it grows to the ovule
(McCormick, 2004); they are
well positioned to mediate events within the pollen tube cytoplasm that result
in changes in the direction of tube extension. Interestingly, because sperm
develop within the pollen tube cytoplasm, the majority of the predicted
N-terminal portion of plasma membrane-localized HAP2 would reside in the
pollen tube cytoplasm and could interact directly with pollen tube cytoplasmic
factors involved in directing tube extension.
Multiple, overlapping chemotropic factors may guide the pollen tube to
ensure optimal efficiency of ovule targeting and fertilization
(Johnson and Lord, 2006).
Therefore, mutations that diminish perception of a single pollen tube-guidance
factor are not expected to completely block ovule targeting. This may explain
why hap2-1 results in a reduction rather than a complete loss of
ovule targeting. The finding that perturbations in the production of
individual female guidance cues such as GABA
(Palanivelu et al., 2003),
ZmEA1 (Marton et al., 2005)
and plantacyanin (Dong et al.,
2005) do not completely block pollen tube guidance or seed
production, supports this view. It will be interesting to test this hypothesis
in the future by constructing plants with defects in production and/or
perception of multiple pollen tube-guidance signals.
HAP2-mediated interactions between sperm and FG are required for fertilization
Approximately half of hap2-1 pollen tubes reach ovules, enter the
micropyle and burst, releasing GUS activity and two sperm into the
degenerating synergid (Fig.
1B). However, hap2 mutant progeny are never recovered
when either hap2-1 or hap2-2 pollen is used to pollinate a
wild-type pistil. This complete transmission block was also observed when the
gcs1 allele was characterized
(Mori et al., 2006). These
results indicate that HAP2 is essential for a step in fertilization
that occurs after sperm have been delivered to the FG by a pollen tube.
Initiation of embryo development and initiation of endosperm development are
both completely blocked in ovules targeted by hap2-1 pollen tubes
(Fig. 2F-H). Furthermore,
gcs1 sperm persist within the degenerating synergid, whereas
wild-type sperm immediately migrate to their egg and central cell target
membranes and fuse (Mori et al.,
2006). Taken together, these results indicate that HAP2 is
probably required for either sperm migration within the FG, or for
binding/fusion of sperm to egg and of sperm to the central cell. Further
analysis of the hap2 fertilization defect using transmission electron
microscopy or live imaging in a system in which hap2 sperm and target
membranes are tagged with fluorescent proteins offers an opportunity to
determine the precise role for HAP2 in fertilization.
Plasma membrane-localized HAP2 could directly mediate gamete-gamete
interactions through its extracellular domain. There is substantial precedence
in animal reproduction for similar sperm-egg interactions; for example, a
sperm-expressed type 1a membrane protein is essential for sperm-egg fusion in
mice (Inoue et al., 2005;
Rubinstein et al., 2006).
Alternatively, ER-localized HAP2 may indirectly mediate fertilization by
regulating the processing or secretion of plasma-membrane proteins or by
regulating calcium levels in sperm.
Insights into the mechanisms of double fertilization
hap2/gsc1 are the only Arabidopsis mutants described so
far in which two sperm are released into the degenerating synergid but
fertilization does not occur. This affords a unique opportunity to clarify
some of the basic mechanisms of double fertilization. Because hap2-1
blocks initiation of both embryo and endosperm development, there must be a
single system in Arabidopsis that mediates interactions between sperm
and egg and between sperm and central cell. Sperm are dimorphic in some
flowering plants, with one sperm type preferentially fertilizing the egg and
the other preferentially fertilizing the central cell
(Roman, 1948;
Russell, 1985). It will be
interesting to determine whether these plants have an additional system to
direct sperm of one type to a specific target or whether HAP2 functions to
mediate specific gamete interactions in these systems.
In wild-type Arabidopsis, only one pollen tube is attracted to
each ovule; attraction of multiple pollen tubes to a single FG would
presumably decrease female fitness and could lead to polyspermy
(Shimizu and Okada, 2000).
Evidence for a repulsive cue that directs supernumerary pollen tubes away from
an ovule that has already been targeted was recently obtained in vitro
(Palanivelu and Preuss, 2006).
Interestingly, the FG mutants feronia and sirene attract
supernumerary pollen tubes; in these mutants, pollen tubes enter the micropyle
but fail to stop growing and burst (Huck
et al., 2003; Rotman et al.,
2003). This shows that pollen tube entry into the micropyle is not
sufficient to trigger production of a repellant. We do not observe
supernumerary pollen tubes on ovules targeted by hap2-1 pollen tubes.
Furthermore, we analyzed 26 ovules targeted by hap2-1 pollen tubes by
staining for GUS activity in synergid cells
(Fig. 4F) and did not find any
that initiated seed development, indicating that supernumerary wild-type
pollen tubes do not fertilize an ovule already targeted by a hap2-1
pollen tube. Taken together, these results indicate that the pollen tube
repellent is produced after the pollen tube bursts but before fertilization,
and suggest that production of a pollen tube repellent may be initiated by FG
perception of a factor present either on the sperm surface or in the pollen
tube cytoplasm.
A new view of sperm cells: no longer passive cargo
The data presented here challenge the assumption that sperm cells are
passive cargo delivered to the FG by the pollen tube. We show that a
sperm-specific gene, HAP2, is required for optimal ovule targeting by
the pollen tube, suggesting that sperm function may impact the growth of the
pollen tube. Recently, it has become clear that despite their compact
chromatin structure, sperm are dynamic cells that express many genes
(Engel et al., 2003;
Engel et al., 2005;
Xu et al., 1999);
HAP2 is the first such gene with a demonstrated function in
reproduction (Mori et al.,
2006). It is likely that more sperm-expressed genes will be
identified that play roles in pollen tube guidance and in fertilization. The
identification of HAP2 presents an opportunity to identify FG-expressed
interacting factors that, together with HAP2, mediate double fertilization, a
process that is crucial for agriculture, but about which we know very little
at the molecular level.
Supplementary material
Supplementary material for this article is available at
http://dev.biologists.org/cgi/content/full/133/23/4761/DC1
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ACKNOWLEDGMENTS |
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