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First published online 28 November 2007
doi: 10.1242/dev.010108
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1 Temasek Life Sciences Laboratory, 1 Research Link, National University of
Singapore, Department of Biological Sciences, 117604, Singapore.
2 Section of Plant Biology, University of California, Davis, 1 Shields Avenue,
Davis, CA 95616, USA.
* Author for correspondence (e-mail: fred{at}tll.org.sg)
Accepted 26 September 2007
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SUMMARY |
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 TOP SUMMARY INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Key words: Pollen, CAF1, Cell cycle, Arabidopsis thaliana
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INTRODUCTION |
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TOPSUMMARYINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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;
Kaya et al., 2001) has a
conserved chaperone activity for chromatin assembly at the DNA replication
fork during S phase. CAF1 targets acetylated histone H3-H4 dimers to newly
synthesized DNA, thus allowing nucleosome assembly
(Polo and Almouzni, 2006). In
mammals, CAF1 is essential for cell viability and consists of the three
proteins p150, p60 and p48. Loss of function of CAF1 activity in mammalian
cells leads to activation of a DNA-damage signaling pathway that slows down
the S phase and arrests the cell cycle
(Haushalter and Kadonaga,
2003). In yeast, the CAF1 assembly complex subunits Cac1, Cac2 and
Cac3 are dispensable for cell viability
(Haushalter and Kadonaga,
2003; Ridgway and Almouzni,
2001), but loss of function of CAF1 causes increased sensitivity
to DNA-damaging stresses (Linger and
Tyler, 2005). Arabidopsis CAF1 contains a core of three
proteins encoded respectively by the p150 homolog FASCIATA1
(FAS1), the p60 homolog FASCIATA2 (FAS2) and the
pRbAp48 homolog MULTICOPY SUPPRESSOR OF IRA1 (MSI1)
(Kaya et al., 2001). The WD40
domain proteins MSI1 and pRbAp46/48 also bind to Retinoblastoma-related
proteins (Ach et al., 1997;
Qian and Lee, 1995;
Rossi et al., 2003). This
presumably regulates activity of the downstream E2F transcription factor,
thereby influencing the gene activation required for entrance into S phase
(Hennig et al., 2005). In
Arabidopsis, reduction of FAS1 and FAS2 activities
upregulates the expression of genes in the DNA-damage response pathway
(Schonrock et al., 2006). In
the absence of CAF1 activity, cells endoreduplicate to higher levels,
suggesting that loss of function of FAS1 or FAS2 affects the
S-G2 and the G2-M transitions in Arabidopsis
(Endo et al., 2006;
Exner et al., 2006;
Kirik et al., 2006;
Ramirez-Parra and Gutierrez,
2007). In parallel to the deregulation of the cell cycle, the loss
of CAF1 function causes a reduction of the heterochromatic fraction
(Kirik et al., 2006), releases
transcriptional gene silencing from endogenous transposons
(Ono et al., 2006) and alters
the pattern of histone acetylation and methylation at promoters of genes
encoding components and regulators of the cell cycle
(Ramirez-Parra and Gutierrez,
2007). It remains unclear whether the vegetative developmental
defects in fas1 and fas2 mutants
(Costa and Shaw, 2006;
Exner et al., 2006) are direct
consequences of the loss of CAF1 on the cell cycle or result indirectly from
the gradual accumulation of epigenetic defects.
In pollen produced from msi1/+ plants, the loss of MSI1 affects
only half of the haploid pollen produced after meiosis. It is thus possible to
investigate the immediate developmental consequence of the loss of CAF1
function using male gametogenesis as a model. Reduced paternal transmission of
msi1 was recorded from the null allele msi1-2
(Guitton et al., 2004),
suggesting the alteration of pollen development by removal of CAF1 function.
Pollen development is characterized by a relatively simple series of events
involving simultaneously cell fate and cell cycle regulation
(McCormick, 2004). We report
that loss of function of CAF1 complex members affects pollen development. Null
alleles of msi1 prevent division in a fraction of pollen. The effect
of msi1 on pollen arrest is synergistically enhanced when CAF1
activity is further compromised in fas1 and fas2 mutants. We
further investigate the fate and differentiation of the pollen in the absence
of CAF1.
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MATERIALS AND METHODS |
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TOPSUMMARYINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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).
The mutant allele msi1-2 (C24 background) was previously described in
the laboratory in F.B. group (Guitton et
al., 2004). The mutant allele msi1-3 (Ler
background) was obtained by V.S. The mutant allele msi1-4
(Col background) was obtained and kindly provided by Gary Drews and Jayson
Punwani (University of Utah). Seeds from qrt1/qrt1 (Ler accession)
were provided by D. Preuss. Homozygote fas1-1 (CS265 En background)
fas1-4 (SAIL_662_D10 Col background) and fas2-4
(SALK_033228, Col background) seeds were obtained from the
Arabidopsis Biological Research Center. The KS22 enhancer-trap line
(C24 accession) expressing the GFP reporter protein was generated in J.
Haseloff's laboratory
(www.plantsci.cam.ac.uk/Haseloff).
Marker lines for cell identity in pollen pDUO1-DUO1-mRFP1 (C24) was obtained
by N. Rotman (Rotman et al.,
2005), AC26 mRFP (C24) was obtained by A. Chaboud (Unite Mixte de
Recherche 9004, Lyon, France) from C24 plants transformed with
pACTIN11-Histone-mRFP1; E1 GFP (Col) was a kind gift from G. Strompen
(Strompen et al., 2005).
ATGEX1 marker line was the kind gift from Sheila McCormick
(Engel et al., 2005). The
marker HTR10-mRFP1 results from a genomic fusion described in Ingouff et al.
(Ingouff et al., 2007).
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A 3050 bp DNA fragment consisting of the upstream region of MSI1 corresponding to the putative promoter until the beginning of exon3 was amplified by PCR using the KOD-plus-PCR kit (TOYOBO, Japan) and cloned into pCR2-1 TOPO vector (Invitrogen, Carlsbad, CA). The final vector pGIInK-promMSI1::MSI1-mRFP1-35S consists of the upstream region of MSI1, its first two exons and introns and the remaining MSI1 cDNA fused to the fluorescent reporter mRFP1. Heterozygous msi1-1/+ mutant BASTA-selected plants were transformed using the Agrobacterium-mediated floral dip method.
Fifteen transgenic lines were obtained, all showing a similar pattern of expression. Maternal transmission of msi1-1 mutation, which is null in the mutant background was completely restored by expression of promMSI1:MSI1-mRFP1 (46.6% transmission of msi1 from seeds produced by msi1/+; promMSI1:MSI1-mRFP1/promMSI1:MSI1-mRFP1 ovules crossed to wild-type pollen, n=250). Plants homozygous for msi1-1 and homozygous for promMSI1:MSI1-mRFP1 were obtained.
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RESULTS |
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TOPSUMMARYINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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We compared our observations of MSI1-mRFP1 expression with the wild-type pattern of chromatin staining by DAPI. The microspore (Fig. 1B) underwent an asymmetric division (PMI), leading to production of bicellular pollen. This comprises the larger vegetative cell and the smaller generative cell initially positioned at the periphery and later engulfed by the vegetative cell (Fig. 1C). The generative cell further divided (PMII) and produced the two sperm cells (Fig. 1D). In contrast to the sperm cells, the large spherical vegetative cell had a larger, less condensed nucleus positioned at the center of the tricellular pollen.
MSI1-mRFP1 was expressed at all stages of pollen development in both cell
types (Fig. 1E-G), as reported
in previous microarray analyses (Honys and
Twell, 2004). The level of MSI1-mRFP1 appeared to increase in the
vegetative cell throughout pollen development, whereas the expression of MSI1
reported by microarrays decreased (see Fig. S1 in the supplementary material).
We favor the idea that the increase of MSI1-mRFP1 signals provides evidence
for new synthesis and accumulation of MSI1-mRFP1 during pollen development. It
is thus unlikely that detected MSI1-mRFP1 was inherited through meiosis. The
mRFP1 fluorescence in the generative cell was weaker than in the vegetative
cell (Fig. 1F). Although the
chromatin of the sperm cells was very compact, the MSI1-mRFP1 signal was
rather low (Fig. 1G).
MSI1 thus appears to be differentially expressed between the
vegetative and generative lineage during pollen development.
Reduced paternal transmission is reported for the allele msi1-2
(Guitton et al., 2004) but not
for the allele msi1-1
(Köhler et al., 2003).
This discrepancy may result from the distinct genetic background of each
allele (C24 versus Col) or from distinct growth conditions. We isolated two
new alleles of msi1 in Ler (msi1-3) and Col
(msi1-4) genetic backgrounds (see Fig. S2 in the supplementary
material). Imperfect insertions of a Ds transposon and of a T-DNA created a
stop codon in the first exon of MSI1 in the alleles msi1-3
and msi1-4. Both alleles do not transmit msi1 maternally and
are presumably null alleles as concluded for the alleles msi1-1 and
msi1-2 (Guitton et al.,
2004; Köhler et al.,
2003).
We measured the paternal transmission in each msi1 allele grown in the same conditions (Table 1). Pollen development takes place after meiosis, and crosses between wild-type ovules and pollen from msi1/+ plants are expected to transmit msi1 in 50% of the offspring if there is full transmission. Paternal transmission was reduced to approximately 36% in every allele (Table 1). Reduced transmission could result from a paternal effect causing reduction of seed germination or seedling viability. The germination rate of wild-type seeds was 98.2% (n=277, s.d.=0.53), hence comparable to the germination rate in seeds from crosses between wild-type ovules and pollen from msi1-1/+ plants (98.0%, n=293, s.d.=0.23). Similarly the survival rate of msi1-1/+ seedlings was not affected (99.6%, n=273, s.d.=0.07). Hence msi1 effect on paternal transmission does not originate from an effect on seed development or on seedlings, and must result from a reduction of male fertility. The reduction of paternal transmission of all four msi1 alleles to 36% indicated a transmission efficiency of 56%. Hence a penetrance of 44% is associated with null mutations in msi1, resulting in defective male gametogenesis in msi1/+ plants. We conclude that msi1 causes defects during pollen development.
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). We analyzed the paternal transmission associated with
the loss-of-function alleles fas1-4 and fas2-4
(Exner et al., 2006;
Ramirez-Parra and Gutierrez,
2007). Paternal transmission of the fas1-4 allele
analyzed from crosses between wild-type ovules and pollen from
fas1-4/+ plants was 49.6% (n=115, s.d.=7.4). Similarly
paternal transmission of fas2-4 was 48.6% (n=140, s.d.=5.2).
According to these data, neither fas1 nor fas2 appear to
affect male gametogenesis. Nevertheless, if we assume that fas1 and
fas2 affect paternal transmission, the associated transmission
efficiency would be of the order of 98%. To test whether the CAF1 pathway is
responsible for the reduced transmission of msi1 null alleles, we
obtained double mutant lines between msi1/+ and loss-of-function
alleles of fas1/+ and fas2/+. Paternal transmission of
msi1 mutant allele was similarly reduced by the introduction of
fas1 and fas2 (Table
1). An additive interaction between fas1 or fas2
and msi1 is predicted to lead to a decrease of msi1
transmission efficiency from 56 to 55% in fas1 or fas2 background.
However, in the fas1 or the fas2 background the transmission
efficiency of msi1 was reduced significantly from 56% to 40%
(Table 1). In conclusion, the
interaction between msi1 and fas1 or fas2 was not
additive but synergistic. Additive interactions between null mutations
indicate that each mutation affects a distinct pathway. However, a synergistic
interaction is observed in combination of partially penetrant alleles of
mutations affecting the same genetic pathway. The synergy observed between
msi1 and fas1 or fas2 on paternal transmission of
msi1 suggests that MSI1, FAS1 and FAS2 act in a
common genetic pathway influencing male gametogenesis.
Loss of MSI1 arrests pollen development
We investigated the developmental origin of the reduced male fertility in
msi1/+ plants. In order to compare directly msi1 and
wild-type pollen development, we used the mutant quartet
(qrt), which produces four microspores remaining associated as a
tetrad (Preuss et al., 1994).
Hence qrt/qrt; msi1-1/+ plants produce tetrads containing
two wild-type and two msi1 pollen grains. Alexander staining for
pollen viability showed 7.3% lethality in msi1 pollen at maturity
(s.d.=1.9; n=800) in comparison with 1.0% in the wild type (s.d.=0.7;
n=500) (see Fig. S3 in the supplementary material). Tetrads of
microspores produced by wild-type and msi1-1/+ plants in
qrt/qrt background could not be distinguished (n=120)
(Fig. 2A). After PMI, in
qrt/qrt, all tetrads consisted of four pollen grains with a
vegetative cell and a generative cell (Fig.
2B). By contrast, a fraction of tetrads in pollen from
qrt/qrt; msi1-1/+ plants contained an arrested microspore
(Fig. 2C). At the mature stage,
in contrast to wild-type pollen tetrads, a fraction of tetrads in
qrt/qrt; msi1-1/+ plants contained an aborted microspore
(Fig. 2D), or a pollen grain
with one (Fig. 2E) or two
(Fig. 2F) nuclei. These results
suggest that the absence of MSI1 causes pleiotropic arrest of pollen
development before PMI, or PMII. The fraction of each class of arrest was
measured in pollen produced by msi1-1/+ plants
(Fig. 3). The percentage of
arrested pollen cumulated with the percentage of aborted microspores amounted
to 14.7%. This value is lower than the 22% abnormal pollen in a population of
pollen from msi1/+ plants, as predicted from the transmission
efficiency of 56%. This suggested that 7.3% of pollen from msi1/+
plants contain two sperm cells and appear morphologically normal, albeit not
functional. These msi1 tricellular pollen grains probably grow a
pollen tube, but the two sperm cells are incompetent for fertilization,
causing ovule abortion. In agreement with this prediction, pollination of
wild-type plants by msi1/+ plants caused a 6% increase in ovule
abortion (see Fig. S4 in the supplementary material). In conclusion, our
observations show that msi1 causes delays and arrests of pollen
development, leading to partial male sterility and reduced paternal
transmission of msi1.
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FACS analyses in young seedlings have shown that loss-of-function mutants
for MSI1, FAS1 and FAS2 lead to a G2/M arrest, presumably as
a result of the activation of the DNA repair pathway following stalling of the
DNA replication fork (Endo et al.,
2006; Exner et al.,
2006; Honys and Twell,
2004; Kirik et al.,
2006; Ramirez-Parra and
Gutierrez, 2007). To investigate the consequence of loss of
activity of CAF1 on cell cycle during pollen development, we measured DNA
content at the early tricellular stage. We compared the DNA content in sperm
cells of wild-type tricellular pollen with the single sperm-like cell in
bicellular pollen from msi1/+;fas1/+ plants. Both
measurements were compared to duo1 pollen single sperm-like cells,
which contain twice the amount of DNA contained in wild-type sperm cells at
the early tricellular stage (Rotman et
al., 2005). The single sperm-like cell in
msi1/+;fas1/+ also contained approximately twice the amount
of DNA in comparison to wild-type sperm cells
(Fig. 4). These measurements
suggest that the loss of CAF1 function delays the cell cycle pace in the
generative cell, preventing the G2-M transition, which leads to PMII in the
wild type.
Cell-fate specification and differentiation is normal in CAF1-deficient pollen
Arabidopsis PMI is coupled to cell-fate specification, leading to
specific expression of vegetative cell
(Twell et al., 1991) and
generative cell (Engel et al.,
2005; Rotman et al.,
2005) markers. The second mitosis is coupled with sperm cell
differentiation, marked by the onset of DNA synthesis
(Durbarry et al., 2005) and
expression of specific genes (Engel et
al., 2005). To further elucidate whether arrests of cell division
in msi1 pollen grain are associated with cell fate changes, we
analyzed cell identities in pollen grains arrested at PMII produced by
msi1/+ plants. These pollen grains contained a large vegetative-like
cell nucleus and a sperm-like cell with condensed DNA
(Fig. 5G). The marker AC26 is
associated with expression of HISTONE 2B-mRFP fusion protein under the control
of the ACTIN-11 promoter and is specifically expressed in wild-type
vegetative cell nuclei (Rotman et al.,
2005) (Fig. 5A,B).
In msi1 pollen arrested at PMII, AC26 was also expressed in the
larger spherical cell in correlation with its vegetative identity
(Fig. 5G,H). We never observed
any pollen grain expressing AC26 in the condensed nucleus of the sperm-like
cell, nor in more than one nucleus, indicating that loss of CAF1 function does
not perturb the vegetative cell identity during pollen development. In
addition, we performed a germination test to estimate whether the vegetative
cell deficient in CAF1 is able to produce a pollen tube. We observed
comparable germination rates between wild-type pollen and pollen from
msi1/+, fas1/+, fas2/+ and the double mutant
combinations, with a slight reduction corresponding to the proportion of dead
pollen recorded for each genetic background (see Fig. S5 in the supplementary
material). Our results support the argument that the vegetative cell
differentiation and function is not affected by the loss of CAF1 function.
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)
(Fig. 5C). The maturing sperm
cells are further marked specifically by the accumulation of the vacuolar
V-ATPase E1 GFP on the outer plasma membrane
(Strompen et al., 2005)
(Fig. 5D) and of the HISTONE3
variant HTR10 in the sperm nucleus
(Ingouff et al., 2007)
(Fig. 5E). Eventually, the
sperm cells specifically express ATGEX1
(Engel et al., 2005)
(Fig. 5F). We observed that all
the markers of sperm cell maturation and identity were expressed only in the
single sperm-like cell in msi1 pollen arrested at PMII
(Fig. 5I-L). The proportion of
pollen with a single sperm cell expressing HTR10-mRFP1 (6%, n=150;
s.d.=2) is similar to the proportion of pollen with a single sperm cell
(Fig. 3). We conclude that all
msi1 single sperm cells express sperm cell fate markers. These
findings indicate that cell fates are not affected in msi1 pollen
grains arrested at PMII, which appear to comprise a vegetative cell and a
single sperm cell.
Pollination with msi1 pollen causes single-fertilization events
In wild-type pollen, the two sperm cells separately fertilize the female
egg cell and the central cell producing the embryo and the endosperm,
respectively (Fig. 6A). The
apparent correct establishment of cell fate in the single sperm-like cell
present in msi1 pollen suggested that it was able to perform
fertilization as a wild-type sperm cell. We suspected that the single sperm
cell in msi1 pollen could fertilize either the egg cell or the
central cell. Crossing wild-type ovules to pollen from msi1/+ plants
produced a low proportion of seeds containing only endosperm (n=20
out of 3600) (Fig. 6B) or an
embryo (n=18 out of 3600) (Fig.
6C). In both cases, these seeds contained residual material from
the unfertilized egg cell or central cell
(Fig. 6B,C), comparable to
those observed in unfertilized ovules (Fig.
6D). We concluded that msi1 pollen with a single sperm
cell probably caused single fertilization events. Alternatively, single sperm
cells may only fuse with one of the female gametes without the fusion of the
parental genetic material (karyogamy), and autonomous development of embryo or
endosperm would follow. If this were the case, the paternal genome would be
excluded and the single embryo or the single endosperm should not express
paternally derived alleles. We tested this hypothesis for the expression of
the endosperm marker KS22 (Ingouff et al.,
2005) (Fig. 6E),
provided by the pollen of msi1/+ plants crossed to wild-type ovules
(Fig. 6F). Seeds with single
endosperm showed expression of the paternally derived endosperm marker KS22
(Fig. 6F, n=7). We
conclude that the single endosperm develops from a central cell fertilized by
the single gamete in msi1 pollen. To establish whether single sperm
cells are able to fertilize the egg cell, we performed ploidy measurements on
seeds containing a single embryo. We observed ten chromosomes at pre-prophase
in single embryo cells (inset in Fig.
6G, n=2). These embryos are thus diploid and have been
produced by fertilization of the egg cell by the single sperm from
msi1 pollen. We conclude that a fraction of msi1 pollen
arrested at PMII delivers a functional single sperm cell equally able to
fertilize the egg cell or the central cell. Similarly, pollination with pollen
from fas1/+ and fas2/+ plants produced seeds containing
either an endosperm or an embryo (0.3%, n=400; and 0.4%,
n=360). This result suggests that CAF1-deficient pollen produces
functional single sperm cells.
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DISCUSSION |
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TOPSUMMARYINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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;
Köhler et al., 2003) and
null msi1 alleles cause production of autonomous endosperm and
abnormal development of endosperm as the other mutants in genes encoding the
subunits of the endosperm Pc-G complex
(Guitton et al., 2004). MSI1
is also likely to be part of Pc-G complexes active during plant vegetative
development (Berger et al.,
2006; Chanvivattana et al.,
2004). Accordingly, partial reduction of MSI1 activity during
vegetative development phenocopies the effect of mutations in vegetative Pc-G
complexes (Hennig et al.,
2005; Hennig et al.,
2003; Katz et al.,
2004; Moon et al.,
2003). However, msi1-null alleles also produce Pc-G
independent phenotypes, including parthenogenesis and sporophytic embryo
lethality, not observed in Pc-G mutants
(Guitton and Berger, 2005;
Guitton et al., 2004).
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;
Grossniklaus et al., 1998).
Other Pc-G genes are expressed during pollen development (see Fig. S1 in the
supplementary material), but their absence does not cause male sterility
(Chanvivattana et al., 2004;
Gendall et al., 2001;
Goodrich et al., 1997;
Wang et al., 2006). FIE is
essential for Pc-G function (Guitton et
al., 2004; Ohad et al.,
1996) and is expressed during pollen development
(Honys and Twell, 2004). If
defects in msi1 pollen depended on Pc-G function, a similar reduction
of paternal transmission should be expected for msi1 and
fie-null alleles. However, fie mutations are fully
transmitted paternally (Guitton et al.,
2004; Ohad et al.,
1996), and we could not observe any pollen defects in
fie/+ mutant (transmission efficiency of the null fie-11
allele from the male is 99.8%; n=800). We thus conclude that
fie null mutations do not affect paternal transmission and that the
effect of msi1 on male transmission does not rely on Pc-G
complexes.
Biochemical studies have demonstrated the potential association of MSI1 to
the Rb-related protein RBR1 (Ach et al.,
1997) but no direct evidence has been provided for a common
function of RBR1 and MSI1 in Arabidopsis. Loss of RBR1 function
alters pollen development. However the phenotypes associated with
rbr1 in pollen are dramatically distinct from msi1 (C.Z. and
F.B., unpublished data) and this difference does not support the association
between RBR1 and MSI1 as the origin of pollen developmental defects in
msi1.
Biochemical evidence has shown that MSI1 is also associated with the two
core subunits of the CAF1 complex, FAS1 and FAS2
(Kaya et al., 2001). G2/M
arrests have been reported in vegetative tissue of fas1 and
fas2 homozygous mutants
(Ramirez-Parra and Gutierrez,
2007). Our study shows the synergy between fas1, fas2 and
msi1 mutations on paternal transmission of msi1 and on
pollen development, strongly suggesting that the defects of msi1
pollen development are caused by the loss of CAF1 function.
The limited penetrance of single mutations in msi1, fas1 and
fas2 may originate from inheritance of wild-type proteins or
transcripts from the microspore mother cell heterozygous for the mutation. In
mammals and Drosophila, CAF1-independent histone chaperone activities
include HISTONE REGULATORY A (HIRA) and ANTISILENCING FACTOR 1 (ASF1), which
are associated directly with the deposition of histones H3 and H4 on newly
synthesized chromatin (Polo and Almouzni,
2006). In Arabidopsis, the limited penetrance associated
with the fas1, fas2 and msi1 mutations might also result
from a redundant activity mediated by the putative HIRA homolog
(Phelps-Durr et al., 2005)
pathway or by a basic function of putative ASF1 homologs
(www.chromdb.org).
Loss of CAF1 function during pollen development arrests the cell cycle but does not alter cell fate
Loss of CAF1 function causes activation of the DNA repair machinery
(Endo et al., 2006;
Exner et al., 2006;
Kirik et al., 2006;
Ramirez-Parra and Gutierrez,
2007) and a decline in CDKA1 activity
(Ramirez-Parra and Gutierrez,
2007). Moreover, the expression of FAS1 is activated at the G1-S
transition by E2F (Ramirez-Parra and
Gutierrez, 2007). Whether the cell cycle and cell fate
deregulation observed in fas1 and fas2 vegetative tissues
(Costa and Shaw, 2006;
Exner et al., 2006) originates
directly from the deficit in CAF1 or results from more indirect epigenetic
deregulations caused by the absence of CAF1 has remained unclear.
Within one to two cell divisions the pollen deficient for MSI1 or FAS1 and FAS2 arrests before the first or the second pollen mitosis. DNA measurements suggested that the cell cycle arrest takes place at the G2-M transition, which is similar to that observed in vegetative tissues in plants and in other species. Hence we propose that loss of CAF1 function causes a cell cycle arrest before the first or the second mitosis during pollen development.
Cell cycle arrests at the G2-M transition would account for the most
prominent phenotypes observed in msi1 pollen. The wild-type sperm
cells reach the mid-S phase, when pollen is shed from the anthers
(Durbarry et al., 2005), and
probably reach the G2-M transition, when they are released into the female
gametes (Friedman, 1999). The
transmission efficiency indicates that an additional fraction of msi1
pollen is unable to transmit msi1 (see Fig. S6 in the supplementary
material). It is thus possible that msi1 pollen in this fraction
contain two gametes that do not reach the G2-M transition when they are
released into the female gametophyte and are unable to fertilize the female
gamete, leading to ovule abortion.
Cell cycle impairment does not prevent differentiation during male gametogenesis
It has become clear that some regulators of cell division take an active
part in cell-fate decisions. In flowering plants, most cells in roots and in
shoots are produced by the activity of meristems, which contain dividing stem
cells (Benfey and Scheres,
2000; Gegas and Doonan,
2006; Scheres,
2001). In the root meristem, cell fate appears to be specified
after the asymmetric division of the stem cell
(Castellano and Sablowski,
2005; Wildwater et al.,
2005). However, the cell fate is established during the G1 phase
(Caro et al., 2007;
Costa and Shaw, 2006) and is
not fixed until the last meristematic division, after which differentiation is
initiated (Berger et al.,
1998). Hence it is possible that cell division influences
cell-fate commitment. The effects of mild alteration of cell cycle regulation
during embryogenesis support the latter hypothesis
(Jenik et al., 2005).
Alteration of CAF1 causes aberrant morphogenesis of trichomes
(Exner et al., 2006) and
alters cell fate in root epidermal cells
(Costa and Shaw, 2006). In
contrast to cell-fate commitment in vegetative tissues, cell-fate
establishment in pollen appears to be largely independent of cell-cycle
deregulation in the msi1 mutants, producing a fraction of bicellular
pollen grains with a functional vegetative cell that delivers a functional
single sperm-like cell. We have shown that msi1 bicellular pollen
correctly expresses cell-fate markers and produces a functional single sperm
cell. Single sperm cells able to fertilize the egg cell are also produced by
the mutant cdka1 (Nowack et al.,
2006). Hence cell fate and cell differentiation appear to be
independent from cell-cycle regulation in pollen development.
Are male gametes specialized for specific fusion with each type of female gamete?
Plumbago zeylanica produces dimorphic sperm cells with specialized
organelle content that specifies the target female gamete
(Russell, 1983;
Russell, 1985). Several
degrees of polymorphism have been reported in other species and in maize lines
harboring supernumerary B chromosomes
(Faure et al., 2003).
Interestingly, B chromosomes are transmitted at higher frequency to the egg
cell (Roman, 1948). Although
such dimorphism was not reported in many species, including
Arabidopsis (Faure et al.,
2003; McCormick,
2004), it is nevertheless possible that isomorphic sperm cells
differentiate to fertilize the egg cell or the central cell exclusively. This
hypothesis was supported by the apparent preferential fertilization of the egg
cell by single sperm cell produced in absence of the cyclin kinase
cdka1 (Iwakawa et al.,
2006; Nowack et al.,
2006). However, the cdka1 single sperm cell may be able
to fertilize the central cell, but this fertilization product may not be
viable and was not detected as such. According to this hypothesis, sperm cell
differentiation would rely on cell-cycle regulation by cdka1.
However, such an event would cause a type of seed abortion, which was not
detected in two independent studies
(Iwakawa et al., 2006;
Nowack et al., 2006). In
contrast to cdka1 pollen, msi1 pollen causes a low
percentage of single fertilization events, leading either to embryo or
endosperm development. The proportion of msi1/+ pollen that delivers
single sperm cells is similar to the proportion of single fertilization
events, suggesting that msi1 single sperm cells are responsible for
the single fertilization events. In addition we have shown that msi1
single sperm cells express the terminal differentiation markers ATGEX1,
accumulate HTR10 as wild-type sperms, and are transported by the pollen tube.
We thus propose that msi1/+ plants produce a fraction of bicellular
pollen with a single fully differentiated sperm cell able to fertilize either
the egg or the central cell.
Thus, the origin of the absence of fertilization of the central cell by
cdka1 pollen remains unclear. The differentiation status of the
cdka1 single sperm cell has not been established and it could be
incompletely functional and able to fertilize only the egg cell. Similarly, it
is still possible that the two sperm cells produced in some msi1
pollen may not reach full competence for fertilization, and one fertilization
may fail and block further development. This could explain why when a single
embryo is produced by msi1 pollen, no autonomous endosperm
development occurs, as reported in cases of single fertilization events by
cdka1 pollen (Nowack et al.,
2006).
Whether in the wild type each of the two sperm cells has an equal capacity
to fertilize either the egg or the central cell for each sperm cell remains
unresolved. Our results rather support the argument that the two sperm cells
are identical, in agreement with the identical morphology of the two sperm
cells, the identical expression of any molecular marker studied to date in
Arabidopsis, and the apparent capacity to fertilize either female
gamete. The results obtained from the study of cdka1 pollen rather
support the opposite idea, which has been so far clearly shown only in the
species Plumbago zeylanica
(Russell, 1983;
Russell, 1985). In vivo
observation of double fertilization in Arabidopsis has been achieved
recently (Ingouff et al.,
2007) and might provide an answer to this long-standing
problem.
Supplementary material
Supplementary material for this article is available at
http://dev.biologists.org/cgi/content/full/135/1/65/DC1
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ACKNOWLEDGMENTS |
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