Molecular Plant Advance Access originally published online on May 21, 2008
Molecular Plant 2008 1(4):611-619; doi:10.1093/mp/ssn016
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Double Fertilization in Arabidopsis thaliana Involves a Polyspermy Block on the Egg but Not the Central Cell
a Department of Biology and Biochemistry, University of Bath, Claverton Down, Bath BA2 7AY, United Kingdom
b School of Biosciences, The University of Birmingham, Edgbaston, Birmingham B15 2TT, United Kingdom
1 To whom correspondence should be addressed. E-mail bssrjs{at}bath.ac.uk, fax 01225 386779, tel. 01225 383437.
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Abstract |
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In animal reproduction, thousands of sperm may compete to fertilize a single egg, but polyspermy blocks prevent multiple fertilization that would otherwise lead to death of the embryo. In flowering plants, successful seed development requires that only two sperm are delivered to the embryo sac, where each must fertilize a female gamete (egg or central cell) to produce the embryo and endosperm. Therefore, polyspermy must be avoided, not only to prevent abnormalities in offspring, but to ensure double fertilization. It is not understood how each sperm fertilizes only one female gamete, nor has the existence of polyspermy barriers been directly tested in vivo. Here, we sought evidence for polyspermy blocks in angiosperms using the polyspermic tetraspore (tes) mutant of Arabidopsis, which allows in-vivo challenge of egg and central cell with multiple male gametes. We show that tes mutant pollen tubes can transmit more than one sperm pair to an embryo sac, and that sperm from more than one pair can participate in fertilization. We detected endosperms but not embryos with ploidies that could only result from multiple fertilization. Our results therefore demonstrate an in-vivo polyspermy block on the egg, but not the central cell of a flowering plant.
Received for publication February 11, 2008. Accepted for publication March 19, 2008.
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INTRODUCTION |
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TOPAbstractINTRODUCTIONRESULTSDISCUSSIONMETHODSFUNDING |
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A defining feature of angiosperms is reproduction by double fertilization. Each pollen grain contains two sperm that are delivered by the pollen tube to the embryo sac, where one fertilizes the egg, forming the zygote, while the other fertilizes the central cell to produce endosperm. This tissue acquires nutrients from the seed parent for transmission to the developing or germinating embryo, and also participates in signalling among seed components (Costa et al., 2004; Berger et al., 2006). Pollen tube guidance and repulsion systems normally ensure that a single pollen tube penetrates each embryo sac (Weterings and Russell, 2004; Dresselhaus, 2006) and therefore each female gamete is presented with a maximum of two sperm. This is in sharp contrast to reproduction in organisms such as echinoderms and fucoid algae that release male and female gametes into water for external fertilization, greatly increasing the number of sperm that may be encountered by each egg. In these taxa, multiple fertilization is prevented by well characterized polyspermy barriers (Schuel, 1984; Brawley, 1992). However, the very low ratio of sperm to female gametes in the angiosperm embryo sac does not necessarily preclude a requirement for a polyspermy block. In some cases, more than one pollen tube enters the ovule, as occurs in heterofertilization in maize, where the egg and central cell are fertilized by sperm from different pairs (Sprague, 1929, 1932; Kato, 2001). Multiple pollen tube entry, described as ‘polyspermy sensu lato’, has also been reported for sunflower and other angiosperm species (Vigfússon, 1970). Even where only two sperm enter the embryo sac, polyspermy barriers might be required to ensure that both egg and central cell are fertilized, as, so far, there is no evidence for mechanisms of active transport of sperm or predestination of each sperm for a particular gamete that are under strict enough control to account for the success of double fertilization (Spielman and Scott, 2008).
In-vitro experiments provide some evidence for a polyspermy barrier on the angiosperm egg, and perhaps also the central cell. Working with calcium- and electrically mediated fusion systems, respectively, Faure et al. (1994) and Kranz et al. (1995) were able to induce single but not multiple fertilization of maize eggs. Maize sperm have also been fused in vitro with central cells, but attempts to induce multiple fertilizations have not been reported (Faure et al., 1994; Kranz et al., 1998). Sun et al. (2000) reported an in-vitro polyspermy block on egg and central cell in tobacco using a polyethylene–glycol-mediated fusion system. However, these experiments, performed in differing media with isolated gametes, may not fully reflect behaviour within the embryo sac. There have been no reports of in-vivo studies designed to explore whether polyspermy blocks operate in double fertilization. Heterofertilization could provide a test bed, but data from these experiments that would unambiguously show the presence or absence of multiple fertilization have not been reported (reviewed by Spielman and Scott, 2008).
In the present work, we tested for polyspermy blocks in a flowering plant using the tetraspore (tes) mutant of Arabidopsis thaliana (Spielman et al., 1997) to challenge both female gametes with extra sperm. In wild-type pollen development, meiosis produces four haploid microspores. Each of these undergoes two rounds of mitosis to produce a pollen grain consisting of a uninucleate vegetative cell, which, on germination, forms the pollen tube, and a pair of haploid sperm cells. tes mutants perform the nuclear divisions of male meiosis but meiotic cytokinesis does not occur, resulting in coenocytic microspores that contain all four haploid nuclei. These nevertheless undergo both pollen mitosis steps to generate pollen grains with up to four pairs of sperm cells. Developmental abnormalities can occur, however, including fusion of the haploid microspore nuclei and loss of nuclei, so that mature grains have a range of sperm numbers and ploidy levels (Spielman et al., 1997). Seeds sired by tes mutants often abort, with a similar phenotype to that generated by crossing a diploid seed parent with a hexaploid pollen parent (2xx6x) in Arabidopsis: embryo arrest around the globular–heart transition, and overproliferation and failure of cellularization in the endosperm (Scott et al., 1998). Abortion of seeds containing an abnormal balance of maternally and paternally contributed genomes has been attributed to parental imprinting (Haig and Westoby, 1991). Imprinting is an epigenetic mechanism, operating on a small proportion of mammalian and plant genes, that allows expression only from the maternal or paternal allele, depending on the locus (Wood and Oakey, 2006; Huh et al., 2007). Therefore, parental genomic imbalance would alter the ratio of active copies of maternally and paternally expressed imprinted genes in the seed. A growing body of evidence demonstrates that imprinting operates in the endosperm (Gehring et al., 2004). Abortion of tes mutant seeds could likewise be due to lethal paternal excess in the endosperm caused by transmission of extra genomes from multiple or polyploid sperm. 2xx4x crosses in Arabidopsis mainly produce seeds with viable paternal excess, characterized by overproliferated endosperm with delayed cellularization, and subsequently heavy seeds containing large triploid embryos; tes mutants likewise produce seeds with these features (Scott et al., 1998).
In the work described below, we found that tes mutant pollen grains could deliver extra sperm pairs to the embryo sac, and that sperm from more than one pair could participate in fertilization. We were able to rescue seeds sired by tes mutants either by raising the ploidy of the seed parent to ameliorate paternal excess, or by combining tes with a DNA methyltransferase1 (met1) mutant, which interferes with the imprinting system (Kinoshita et al., 2004; Jullien et al., 2006)—thus confirming that seed abortion in tes mutants is most likely related to imprinting, and demonstrating that tes mutant sperm do not have functional abnormalities other than polyploidy. We karyotyped seedlings and developing endosperms from seeds sired by tes mutants, and found endosperms but not embryos with chromosome numbers that could only result from multiple fertilization. Our results are therefore consistent with a polyspermy barrier on the egg but not the central cell of a flowering plant.
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RESULTS |
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TOPAbstractINTRODUCTIONRESULTSDISCUSSIONMETHODSFUNDING |
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tes-3 Mutant Pollen Grains Contain Multiple Sperm Pairs at a Range of Ploidy Levels
We first sought to establish whether tes mutant pollen is useful as a test system for challenging eggs and central cells with multiple sperm in vivo. Our previous phenotypic analysis of tes mutants focused on the tes-4 allele in the Ws2 accession (Spielman et al., 1997). Of the four alleles we tested, however, tes-3 in Ler produced the highest mean number of sperm per pollen grain, and therefore should provide the greatest opportunity for polyspermy. In the present work, we confirmed that, like tes-4 (Spielman et al., 1997), tes-3 pollen contains multiple pairs of sperm at different ploidy levels (Figure 1). We found that 42% of tes-3 pollen grains contained more than two sperm, with the most frequent class of grains with extra sperm having four (34%, n = 100) (Figure 1A and 1B). Sperm cell ploidy (see Methods) varied between 1x and 4x, with 89% of pollen grains containing sperm of greater than 1x (Figure 1C). The complete distribution of sperm numbers and ploidies we observed in tes-3 pollen grains is shown in Figure 1D.
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Seventy-two percent of seeds from self-pollinated tes-3 mutants aborted (Figure 2A and 2B). Control crosses between wild-type plants of different ploidies showed that, like the C24 accession (Scott et al., 1998), Ler plants tolerate an endosperm genomic ratio of 2m:2p, generated by 2xx4x crosses, but not 2m:3p, generated by a 2xx6x cross (Figure 2A and 2B). Seeds fathered by tes-3 mutants have a mixture of normal and paternal excess phenotypes (Figure 2C). Taken together, this suggests that lethal paternal excess in tes-3 could result from fertilization of the central cell either by a single sperm of high ploidy (
3x, monospermy) or by multiple sperm of lower ploidy (additively 3x, polyspermy).
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Abortion of tes-3 Mutant Seeds is Rescued by Loss of MET1 Function
We first wanted to investigate whether multiple tes sperm are a true proxy for wild-type sperm: otherwise, generalizations based on the behaviour of tes mutants may be invalid. To rule out the possibility that abortion in tes mutants is due to sperm defects, we attempted to rescue seeds sired by tes-3 mutants using a loss-of-function mutation in the MET1 gene. met1 mutations have been shown to cause hypomethylation of the genome (Kankel et al., 2003). DNA hypomethylation using an antisense construct to MET1 appears to confer ‘maternal’ characteristics on the sperm genome: so, for example, crossing a wild-type seed parent with a pollen parent carrying a MET1 antisense construct generates small seeds with endosperms that cellularize early—both features of maternal excess (Adams et al., 2000; Spielman et al., 2001). It is well established that silencing of imprinted genes in flowering plants, as well as in mammals, involves cytosine methylation (Wood and Oakey, 2006; Huh et al., 2007). Furthermore, it has been demonstrated for several imprinted loci in Arabidopsis that seeds sired by met1 mutants ectopically express the paternally derived alleles of genes that normally are only expressed from maternal alleles (Kinoshita et al., 2004; Jullien et al., 2006). Therefore, we predicted that tes sperm developed in a hypomethylated background would not have a lethal paternalizing effect on endosperm. However, if seed lethality is due to factors other than paternal excess, hypomethylation would not be expected to rescue.
The alternatives were tested using double mutants between tes-3 and the met1-9 T-DNA insertion mutant. These produced pollen with a tes-3 mutant phenotype, including sperm numbers similar to the tes-3 allele in the original background (tes-3: two sperm, 59%, four sperm, 36%, six sperm, 4%, eight sperm, 1%, n = 100; tes-3 met1-9: two sperm, 54%, four sperm, 40%, six sperm, 6%, eight sperm, 0%, n = 50); 
2 = 0.534, P = 0.766). The majority of seeds from crosses between wild-type seed parents and tes met1 pollen parents were plump and viable (Figure 2D, left), whereas seed sired by tes-3 MET1 siblings showed levels of abortion similar to the 2xxtes-3 control cross (Figure 2D, right). This demonstrates that sperm abnormalities are unlikely to be the cause of abortion in tes mutant seeds, as there is no obvious mechanism by which hypomethylation could compensate for changes such as loss of chromosomes or developmental arrest that might occur in coenocytic tes pollen grains.
The Multiple Sperm Pairs in tes Pollen Are Available for Fertilization
tes mutant pollen tubes germinated in vitro often carry more than one sperm pair (tes-4, Spielman et al., 1997; tes-3, Figure 3A). tes-3 pollen tubes containing more than two sperm occurred at a frequency of 35% (n = 57), with the majority harbouring two pairs of sperm, and occasionally three pairs. To determine whether extra sperm pairs can enter the embryo sac and fertilize the egg and central cell, we looked for evidence that the female gametes within an individual embryo sac were fertilized by sperm from two different germ units (‘uncoupling’).
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In the first approach, we produced plants homozygous for tes-3 and either hemi- or homozygous for a reporter of embryo and endosperm fertilization comprising the Arabidopsis MINISEED3 (MINI3) promoter fused to β–GLUCURONIDASE (GUS) (Luo et al., 2005). In control crosses, wild-type plants were fertilized with pollen from tes-3 plants homozygous for proMINI3-GUS. GUS activity was observed in both the embryo and endosperm in 95% of seeds (n = 54) (Figure 3B, left), while the remaining 5% had no staining in either fertilization product (not shown). In contrast, when wild-type plants were fertilized with pollen from tes-3 plants hemizygous for the reporter construct, GUS activity could be detected exclusively in the endosperms in 10% of seeds (n = 82) (Figure 3B, middle). Rarely, GUS activity was detected exclusively in the embryo and suspensor (Figure 3B, right). These outcomes indicate that, in some cases, the egg and central cell were fertilized by sperm from different germ units—one carrying the fertilization reporter and the other without the reporter. If polyspermy occurs, the frequency of embryos and endosperms with differential staining would under-represent the frequency of uncoupling, as sperm from more than one pair could have the same genotype with respect to the reporter.
We next sought confirmation of uncoupling by exploiting the relationship between seed size and m:p ratio in the endosperm, as seeds with viable paternal excess (containing 2m:2p endosperms) are larger than seeds from balanced crosses (2m:1p) (Scott et al., 1998). We previously showed that tes-4 mutant sperm within individual pairs have the same DNA content (Spielman et al., 1997), and this was also observed for tes-3 (not shown). Fertilization of egg and central cell by sperm of different ploidies, and hence from different sperm pairs, would disrupt the normal relationship of seed size and embryo ploidy to produce normal sized seeds (with 2m:1p endosperms) containing high ploidy embryos (> 2x), or large seeds (2m:2p) with normal ploidy embryos (2x). Wild-type 2xxtes-3 crosses produced a mixture of shrivelled and plump seeds (Figure 3C, left), and the latter were easily separated into two size classes (Figure 3C, middle). Ploidy analysis of seedlings by flow cytometry showed that 27% of the normal-sized seeds contained a high ploidy (3x) embryo and 32% of the large seeds contained a normal ploidy (2x) embryo (Table 1). This is in contrast to control pollinations where uncoupling cannot occur (Figure 3C, right), in which embryo ploidy was always correlated with seed size (Table 1). We also found that 2xxtes-3 crosses yielded a small proportion (3%) of 4x offspring. A 4x embryo would have required fertilization of the 1x egg by a single 3x sperm (given our finding that there is a polyspermy block on the egg; see below). Such a contribution into the central cell would result in the invariably lethal 2m:3p ratio, suggesting that uncoupling must occur to produce these seeds. Taken together, the uncoupling experiments demonstrate that multiple sperm enter the embryo sac and are available for fertilization.
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The Egg is Resistant to Multiple Fertilization
To investigate whether the egg is susceptible to multiple fertilization, we assayed the ploidy of progeny of tes mutant fathers to look for seedlings with chromosome numbers that were greater than could be achieved through monospermy. To increase the range of embryo ploidies that could be observed, we used crosses that increase the frequency of viable seed by raising maternal ploidy (4xxtes-3) or, according to our model, by maternalizing sperm (2xxtes-3 met1-9). Replacing a diploid with a tetraploid seed parent reduced the abortion frequency in crosses with a tes-3 father from 72% (Figure 2B) to 24% (Figure 4A and 4B) (n = 124). Analysis of plants grown from 4xxtes-3 seeds revealed a range of ploidy levels (3x to 6x) that could be generated through monospermy (2x egg fertilized by a 1x, 2x, 3x, or 4x sperm) or polyspermy, but no plants of 7x or 8x, which would provide unambiguous evidence for multiple fertilization of the egg (Figure 4C). This is consistent with the operation of an egg polyspermy barrier.
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However, since Arabidopsis plants of very high ploidy, such as 7x and 8x, have not been reported, it was possible that seeds containing such embryos are inviable and may have been lost among the aborted seeds from 4xxtes-3 crosses. We therefore examined the ploidy range of plants grown from 2xxtes-3 met1-9 seeds, which had only 3.5% abortion (n = 109). We again found the full range of monospermic ploidy levels, in this case 2x to 5x as the egg was haploid, and again no individuals with ploidy levels requiring polyspermy (Figure 4D). Taken together, this evidence suggests the operation of a polyspermy block on the egg of Arabidopsis.
In these experiments, we did not expect polyspermy per se to cause embryo abortion. In most animals, polyspermy results in formation of multi-polar or supernumerary mitotic spindles in the zygote due to transmission of extra centrioles from the sperm, resulting in aberrant divisions and death of the embryo (Schuel, 1984; Navara et al., 1994). Likewise, in fucoid algae, sperm contribute centrioles to the zygote, and multi-polar spindles followed by abnormal cytokinesis have been observed in polyspermic zygotes of Fucus distichus (Nagasato et al., 1999). However, angiosperms lack centrosomes (Lloyd and Chan, 2006), and therefore a polyspermic zygote would not die for the same reason that an animal or algal zygote would fail. Furthermore, polyploidy is no barrier to plant development (Bennett, 2004). Therefore, we would expect to recover polyspermic embryos if they were ever formed.
The Central Cell is Receptive to Multiple Fertilization
Next, we investigated whether entry of multiple sperm pairs into the embryo sac leads to multiple fertilization of the central cell. The endosperm karyotype was directly visualized in individual seeds produced by 2xxtes-3 crosses, at a stage before those with lethal paternal excess collapse and die, using chromosome spreading with DAPI labelling (Figure 4E) or Fluorescence In Situ Hybridization (FISH) (Figure 4F). In this cross, a chromosome number of 30, generated by fertilization of the 2x (10 chromosome) central cell with a 4x (20 chromosome) sperm, is the highest achievable through monospermy. However, endosperms containing 35, 40, or 45 chromosomes were observed, at a combined frequency of 9.1% (n = 144) (Figure 4E and 4F; Table 2), indicating multiple fertilization of the central cell. This figure is likely to be an underestimate of the potential polyspermy rate. Only about 35% of tes pollen grains could contribute enough chromosomes to the central cell to provide unambiguous evidence for multiple fertilization (i.e. at least 25 chromosomes after one sperm is subtracted for the embryo, based on the data presented in Figure 1D); and this percentage is likely to be diminished further by the failure of some tes sperm in a pollen grain to reach the embryo sac (data not shown), and the frequent contribution of 10 or more chromosomes to the embryo (Figure 4C and 4D). Therefore, based on our observations of the distribution of sperm number and ploidy in tes-3 grains, at least 26% (i.e. 9.1%/35%) of the central cells that were exposed to multiple sperm showed evidence of polyspermy, but the actual figure is likely to be greater. For the reasons mentioned above, and also because we could not distinguish whether endosperms with chromosome numbers between 20 and 30 were generated by single or multiple fertilization, our data represent a minimum estimate of the polyspermy rate in Arabidopsis endosperm when the central cell is exposed to multiple sperm.
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DISCUSSION |
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TOPAbstractINTRODUCTIONRESULTSDISCUSSIONMETHODSFUNDING |
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Here, we provide evidence for a polyspermy block in vivo on the egg but not the central cell of Arabidopsis thaliana. This could form part of the mechanism to ensure that each female gamete is fertilized by one and only one sperm. After delivery to the embryo sac, sperm appear to use an actomyosin system to travel toward the egg and central cell (Weterings and Russell, 2004). However, there is no known mechanism for strictly ensuring that each sperm migrates toward or fuses with only one gamete (reviewed by Spielman and Scott, 2008). Intriguingly, the cdc2a mutant of Arabidopsis produces pollen bearing a single unreduced gamete, which accomplishes single fertilization—always of the egg (Nowack et al., 2006). This raises two possibilities: either the first male gamete to leave the pollen tube (which, in the case of the cdc2a mutant, is the only gamete) is always transported to the egg while the second migrates toward the central cell, or else both sperm initially compete for the egg but only one gains entry. The latter alternative would require a polyspermy block on the egg. However, these models are challenged by the recent finding that msi1 mutants in Arabidopsis produce a small proportion of pollen grains containing a single haploid gamete expressing markers of sperm differentiation that appears to fertilize egg and central cell with equal frequency (Chen et al., 2008). Clearly, more information about the mechanisms of fertilization will be needed to understand how double fertilization is so reliably achieved.
Heterofertilization of egg and central cell by sperm from different pairs was first described in maize nearly 80 years ago (Sprague, 1929); in this case, the multiple sperm pairs are delivered by two different pollen tubes that enter the same embryo sac. Heterofertilization also provides an opportunity to study polyspermy blocks in vivo. The recovery of polyploid seedlings would indicate multiple fertilization of the egg. Although Sprague (1929) did not consider this possibility, and did not supply directly relevant data, his genetic analysis of plants derived from heterofertilized kernels showed segregation ratios consistent with diploidy, suggesting that maize could operate a polyspermy block in vivo as well as in vitro. Maize endosperm is extremely intolerant of paternal excess (Cooper, 1951) and, therefore, multiple fertilization of the central cell should result in seed abortion; however, abortion frequencies are not generally recorded in the heterofertilization literature. Our results suggest that revisiting heterofertilization from the viewpoint of polyspermy blocks will add to our understanding of the control of double fertilization.
We also extended the findings for maize (Lin, 1984) that parental genome balance, while crucial for the endosperm, does not directly affect the embryo. We were able to recover 4x seedlings from seeds with a tes-3 mutant pollen parent, which would have had triple the normal paternal (relative to maternal) chromosome dosage, while this level of paternal excess in endosperm is invariably lethal (Scott et al., 1998). These data therefore add to the evidence that parental imprinting operates in the endosperm, not embryo, of flowering plants (Gehring et al., 2004).
In most animal species, supernumerary sperm are prevented from entering the egg cytoplasm; however, some taxa (such as birds and amphibians) operate a ‘physiological’ polyspermy barrier, allowing multiple sperm to fuse with the egg but only one to merge with the egg pronucleus (Wong and Wessel, 2006). It remains to be tested whether the polyspermy block on the Arabidopsis egg operates at the level of plasmogamy or karyogamy.
Polyspermy poses a special problem for apomictic species, which produce seeds containing asexual embryos. Although the egg is not fertilized, in the majority of apomicts, the embryo is nurtured by a sexual endosperm (Nogler, 1984). Apomicts undergo male meiosis and produce haploid sperm, but the central cell is generally unreduced. This potentially generates a 4m:1p ratio in the endosperm, which is typically lethal in sexual species (Haig and Westoby, 1991). Accordingly, some apomicts have modified aspects of reproductive development, presumably to restore a normal genomic balance in endosperm, such as by fertilizing the central cell with two sperm, or by forming a central cell with only one unreduced polar nucleus, which is fertilized by a single sperm (reviewed by Nogler, 1984; Spielman and Scott, 2008). In the former case, multiple fertilization of the central cell is required, while, in the second, it must be prevented. Other apomictic species tolerate deviations from 2m:1p in endosperm, and, in these, it may also be important to prevent the ‘spare sperm’ that was not used to fertilize the egg from fusing with the central cell. Further study of fertilization in apomicts is needed to understand how multiple fertilization of the central cell is avoided or promoted, and also shows that polyspermy must be taken into account in strategies to transfer apomixis to crop plants.
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METHODS |
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TOPAbstractINTRODUCTIONRESULTSDISCUSSIONMETHODSFUNDING |
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Plant Stocks
2x Col and 2x Ler were supplied by the European Arabidopsis Stock Centre (NASC) (Nottingham, UK). 4x Col, 6x Col, and 4x Ler were gifts of Luca Comai. We generated 6x Ler by crossing a 4x Ler seed parent with a tes-3 (Ler) pollen parent, and selecting phenotypically wild-type, 6x progeny. tes-3 is described by Spielman et al. (1997). tes-3 mutants were confirmed by karyotyping to be 2x before use in the experiments described here. The met1-9 mutant (Salk_076522), which harbors a T-DNA insertion in the first exon, was generated by SIGnAL (Alonso et al., 2003) and obtained from NASC. The proMINI3-GUS reporter (Luo et al., 2005) was kindly supplied by Fred Berger. For crosses between plants, stamens were removed from buds before anther dehiscence, and pollen was applied to the stigma 2 d later.
Imaging of Pollen Nuclei
Freshly opened flowers were vortexed briefly in 1 ml of 25% (w/v) sucrose to release pollen grains. The flowers were removed and pollen was pelleted by centrifugation for 2 min at 10 000 g, then resuspended in 100 µl of 25% (w/v) sucrose containing 2.5 µg ml–1 4',6-diamidino-2-phenylindole (DAPI), 0.1% Triton X-100 and incubated at 20°C for 20 min. DAPI-labelled sperm nuclei were visualized with a Nikon Eclipse 90i microscope equipped with epifluorescence. Images were captured in JPEG format with a Nikon Digital Sight DS-1QM/H cooled CCD camera. For visualization of nuclei in pollen tubes, pollen was germinated as in Spielman et al. (1997) and labelled with DAPI.
Sperm Ploidy Analysis
Pollen was labelled with DAPI as above and visualized with a Nikon TE2000 fluorescence microscope with a Hamamatsu ER-ORCA CCD camera and a Sutter Lambda 10-2 controller and filter wheel. Images were captured using Perkin Elmer Ultraview software. Areas of sperm nuclei were measured by drawing around the perimeter with Visilog 5.0 software (Noesis, France). Mean areas of 50 sperm nuclei from wild-type 2x, 4x, and 6x Col plants were used as controls. Areas of 210 sperm nuclei from tes-3 pollen were measured, and their ploidy was estimated based on similarity to areas from control sperm of known ploidy.
Imaging of Seeds
Developing seeds were cleared in chloral hydrate:water:glycerol (8w:3v:1v) and visualized with a Nikon Eclipse 90i microscope using differential contrast optics. Images were captured in JPEG format with a Nikon Digital Sight DS-U1 camera. Mature seeds were photographed with a Nikon SMZ1500 stereomicroscope using a Nikon Digital Sight DS-U1 camera.
GUS Staining
To visualize proMIN3-GUS activity, seeds were stained as in Tiwari et al. (2006). The reaction was stopped with 70% ethanol, then seeds were cleared in chloral hydrate:glycerol:water as above, and photographed with a Nikon 90i Eclipse microscope under differential contrast optics with a Nikon Digital Sight DS-U1 camera.
Seedling Ploidy Analysis
Ploidy of seedlings was analyzed using flow cytometry performed by Plant Cytometry Services (Schijndel, the Netherlands). For karyotyping of endosperm, chromosome spreads were prepared as in Armstrong et al. (2001) with the addition of a pre-treatment to accumulate mitotic figures: seeds were dissected from siliques and incubated in 2.5 mM 8-hydroxyquinoline, 100 µM oryzalin, 100 µM colchicine at r.t. for 4 h, then fixed as described. Chromosome spreads were labelled with DAPI or, for FISH, hybridized with digoxigenin-labelled pAL1 probe containing a 180-bp pericentromeric repeat (Martinez-Zapater et al., 1986) and detected as described by Armstrong et al. (2001).
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FUNDING |
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TOPAbstractINTRODUCTIONRESULTSDISCUSSIONMETHODSFUNDING |
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Nikon UK Ltd support the Nikon and University of Bath Imaging Suite (NUBIS) and the Biotechnology and Biological Sciences Research Council (BBSRC), UK, funded M.S.
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Acknowledgements |
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We thank Laurence Hurst, Thomas Dresselhaus and Hugh Dickinson for advice on the manuscript, Karel Riha for improvements to the chromosome squash protocol, and the Salk Institute Genomic Analysis Laboratory for providing the sequence-indexed Arabidopsis T-DNA insertion mutant met1-9.
No conflict of interest declared.
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