1 Laboratoire de Reproduction et Développement des Plantes, UMR 5667,
Ecole Normale Supérieure de Lyon, 46 Allée d'Italie, F-69364
Lyon, Cedex 07, France
2 Plant Research Department, PRD-301, Risø National Laboratory, PO Box
49, DK-4000 Roskilde, Denmark
3 ZMBP Center of Plant Molecular Biology, Developmental Genetics,
University of Tübingen, Auf der Morgenstelle 3, D-72076 Tübingen,
Germany
4 Carnegie Institution of Washington, Department of Plant Biology, 260 Panama
Street, Stanford, California 94305, USA
5 Laboratoire de Biologie des Semences, UMR INRA/INA-PG, Route de Saint-Cyr,
78026 Versailles Cedex, France
* Author for correspondence (e-mail: frederic.berger{at}ens-lyon.fr)
Accepted 6 September 2002
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SUMMARY |
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Key words: Arabidopsis thaliana, Endosperm, Seed, Cytokinesis, SPÄTZLE
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INTRODUCTION |
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The prominent role played by the microtubule cytoskeleton in this process
is well supported by genetic evidence. Mutations in the Arabidopsis
PILZ genes encoding the microtubule folding cofactor complex
(Steinborn et al., 2002)
severely disrupt microtubule organisation and result in a complete block of
mitosis and cytokinesis both in embryo and endosperm
(Mayer et al., 1999
). Other
mutations, such as tonneau1 and fass/tonneau2, more
specifically affect organisation of cortical microtubules: fass/ton2
embryos, for example, lack both the cortical array of parallel microtubules
and the PPB (Traas et al.,
1995
; McClinton and Sung,
1997
). As a consequence, fass/ton2 cells undergo
unordered expansion in all dimensions and form oblique irregular cell walls
very unlike the well-ordered cell files that are representative of
Arabidopsis embryos (Torres-Ruiz
and Jürgens, 1994
; Traas
et al., 1995
).
Several mutations that affect cytokinesis in the Arabidopsis
embryo have been isolated, including knolle and keule
(Mayer et al., 1991).
KNOLLE encodes a member of the syntaxin family, a class of
membrane-bound receptors required for docking and fusion of vesicles at the
target membrane (Lukowitz et al.,
1996
). The KNOLLE protein only accumulates at mitosis and
localises to the plane of division, indicating that its specific role is to
mediate membrane fusion at the cell plate
(Lauber et al., 1997
). The
keule (Assaad et al.,
1996
) and hinkel
(Strompen et al., 2002
)
mutants display cytokinesis defects during embryo development similar to those
in knolle. The KEULE gene encodes a member of the Sec1
family (Assaad et al., 2001
).
SEC1 proteins regulate vesicle trafficking and interact with syntaxins. E.
coli-expressed KNOLLE binds KEULE from plant extracts in vitro,
suggesting that the two proteins interact directly during cytokinesis. In
support of this, the knolle keule double mutant displays a synthetic
embryo-lethal phenotype, with cytokinesis completely prevented
(Waizenegger et al., 2000
).
The HINKEL gene encodes a kinesin-like protein apparently involved in
reorganisation of phragmoplast microtubules
(Strompen et al., 2002
).
Additional mutations in genes non-allelic to KNOLLE, KEULE and
HINKEL but with similar embryo-lethal phenotypes have recently been
isolated. These mutants include runkel
(Nacry et al., 2000
), open
house (allelic to findling; W. Lukowitz and C. Somerville,
unpublished) (Nacry et al.,
2000
) and a novel allele of pleiade, first isolated as a
root-specific cytokinesis mutant (Hauser
and Bauer, 2000
). The functions and products of the mutated genes
are not known.
In flowering plants the pollen delivers two male gametes that fuse with the
egg cell and the central cell present in the ovule. The ensuing fertilisation
product of the central cell develops as the endosperm. In Arabidopsis
the early endosperm development is characterised by a series of synchronised
nuclear divisions that are not followed by cytokinesis
(Mansfield and Briarty,
1990a). This results in the formation of a large multinucleate
cell, a syncytium. The syncytial Arabidopsis endosperm contains three
distinct domains (Brown et al.,
1999
). In this study we refer to these as the micropylar
endosperm, surrounding the embryo at the anterior pole (MCE), the peripheral
endosperm (PEN) and the chalazal endosperm (CZE) at the posterior pole,
reflecting the arrangement along the anterior-posterior axis defined by the
site of delivery of the sperm (anterior). While nuclei in the MCE share a
common mass of cytoplasm, each nucleus in the PEN is associated with its
individual cytoplasm surrounded by a dense array of microtubules in a
structure termed a nucleocytoplasmic domain (NCD)
(Brown et al., 1999
). In MCE
and PEN pseudo-synchronous mitoses take place at different paces
(Boisnard-Lorig et al., 2001
).
At the globular stage of embryogenesis, the syncytial endosperm contains
approximately 100 nuclei (Stage VIII). At this stage, the formation of cell
walls that partition nuclei and cytoplasm into individual cells is initiated
in the MCE. This process, termed cellularisation, was reported to proceed as a
wave across the PEN towards the CZE
(Mansfield and Briarty, 1990b
;
Brown et al., 1999
).
Cellularisation establishes one endosperm cell layer and precedes further cell
division. In maize and in barley, complex microtubule rearrangements take
place prior to endosperm cellularisation. During cellularisation so-called
adventitious or cytoplasmic phragmoplasts are observed at the junctions of
opposing ends of radial microtubules that encircle each nucleus
(Brown et al., 1994
). Similar
adventitious phragmoplasts are seen during Arabidopsis endosperm
cellularisation (Brown et al.,
1999
). Recently, detailed studies of endosperm cellularisation in
Arabidopsis have confirmed that cell wall formation is mediated by
specialised types of phragmoplasts and cell plates
(Otegui and Staehelin, 2000b
).
The syncytial-type cell plates go through a series of maturation steps that
are similar, but not identical, to those observed in conventional cytokinesis
(Otegui and Staehelin, 2000a
).
While cytological descriptions of endosperm cellularisation thus indicate its
unique features, evidence for the accumulation of both KNOLLE
(Lauber et al., 1997
) and the
dynamin-like protein ADL1A (Otegui et al.,
2001
) in both somatic- and syncytial-type cell plates hints at
shared components in the two processes.
We report in vivo observation of cellularisation in the PEN of Arabidopsis and show that it follows nuclear division, as in conventional cytokinesis. We evaluate the differences and similarities between conventional cytokinesis and endosperm cellularisation using a genetic approach. We analyse the effect on endosperm cellularisation of six Arabidopsis mutations that affect cytokinesis in the embryo. This analysis shows that most embryo cytokinesis-defective mutants display similar defects in endosperm cellularisation. In addition, we characterise mutant alleles of a novel gene, SPÄTZLE, in which endosperm cellularisation is defective but cytokinesis is normal in the embryo and in plants homozygous for the mutation.
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MATERIALS AND METHODS |
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Feulgen staining and confocal microscopy
Individual Arabidopsis thaliana siliques were opened with two
shallow longitudinal cuts on either side of the false septum. Siliques were
stained with Schiff's reagent (Sigma) and embedded in LR-white (Sigma)
according to the method of Braselton et al.
(Braselton et al., 1996). All
mutant lines were initially propagated as heterozygotes and produced siliques
that contained both wild-type seeds and seeds that displayed the mutant
phenotype. Seeds that originated from individual siliques were isolated in
each preparation in order to be able to compare the mutant and wild-type
development at corresponding stages. Confocal microscopy was performed using a
Zeiss LSM-510 microscope using the 488 nm excitation line of an argon laser
and a long pass emission filter at 510 nm.
Time-lapse imaging of cellularisation
Seeds of an endosperm marker line from the enhancer trap collection of J.
Haseloff were placed in a small moist chamber as described previously
(Boisnard-Lorig et al., 2001).
Imaging of the GFP fluorescence (488 nm excitation; 510-550 nm emission) was
performed for 12 to 24 hours using a time-lapse automated recording of
confocal sections every 10 minutes. We used a x 16 objective n.a. 0.7.
with oil immersion. 1024x1024 pixel confocal sections were processed
using Photoshop 5.0 (Adobe). Movies were produced with Metamorph.
Genetic mapping
The homozygous mutant line DRU42 in WS ecotype was crossed to wild-type
Landsberg erecta. F2 seeds from F1 progeny
heterozygous for the DRU42 mutation were used to generate the mapping
population. The phenotype of individual F2 plants was determined
and the wild-type plants were used to investigate recombination events with
polymorphic markers (CAPS and SSLP). 49 plants were analysed for three markers
per chromosome.
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RESULTS |
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To perform a detailed analysis of the timing and dynamics of endosperm
cellularisation we have taken advantage of an Arabidopsis enhancer
trap line that expresses a variant of the green fluorescent protein, mGFP5
(Haseloff et al., 1997),
uniformly in the developing endosperm
(Boisnard-Lorig et al., 2001
).
mGFP5 is retained in the endoplasmic reticulum and during cytokinesis
fluorescence from mGFP5 accumulates at the forming cell plate
(Haseloff, 1999
). This
labelling permits in vivo observation, using confocal microscopy, of endosperm
at the transition between the stages VIII and IX when PEN cellularisation
takes place. At the end of stage VIII, cellularisation is complete at the
micropylar pole around the embryo but the PEN remains syncytial
(Fig. 1A, 0 minutes; MCE shown
only in supplementary material). At this point the NCDs remain unchanged for
an extended period, without any signs of cell wall formation or division. In
the data presented here, this period is more than 3 hours
(Fig. 1A,B). In multiple
observations, each with a duration greater than 6 hours we did not observe any
extension into the PEN of the cell walls present in the MCE. At the transition
to stage IX, simultaneous nuclear divisions are initiated in the micropylar
mitotic domain and propagate rapidly as a wave across the PEN
(Fig. 1C; total duration
approximately 40 minutes, the anterior pole is toward the top of the figure)
as reported for earlier developmental stages
(Boisnard-Lorig et al., 2001
).
In the already cellularised MCE, GFP fluorescence is detected in a small band
between sister nuclei, about 15 minutes after karyokinesis (see movie:
http://dev.biologists.org/supplemental/).
This band expands centrifugally as in conventional cytokinesis and delineates
the nascent cell wall between the two daughter cells. In the syncytial PEN a
GFP signal is first detected between post mitotic nuclei that still form a
distinguishable pair, 30-40 minutes after telophase
(Fig. 1D). Over a period of 60
minutes after karyokinesis, cellularisation becomes apparent throughout the
peripheral endosperm (Fig. 1E).
It was often difficult to observe more precisely the extension of the band of
GFP fluorescence between individual nuclei because of growth of the seed that
displaced the observed nuclei out of the focal plane. Initiation of PEN
cellularisation was, however, repeatedly (n=13) observed immediately
after the eighth wave of mitosis that crosses the endosperm from the anterior
to the posterior pole. Cellularisation is completed without any further
divisions occurring (Fig. 1F).
Fortuitous observation of a developing seed fixed at a point during the
advancement of the eighth mitotic round revealed further details of the first
steps of cellularisation. Fig.
1G-J are periclinal views of dividing nuclei positioned along the
posterior to anterior axis. The mitotic stage seen mostly frequently at the
posterior (i.e. the more recently produced) is metaphase
(Fig. 1G). Toward the anterior
pole anaphase (Fig. 1H) and
telophase (Fig. 1I) are
observed successively, immediately followed by formation of a structure
resembling a cell plate between separating sister nuclei
(Fig. 1J). It thus appears that
cellularisation follows a series of events initiated in conjunction with a
synchronised division, where a cell plate is first formed between sister
nuclei, followed rapidly by assembly of cell walls between non-sister nuclei
throughout the PEN, resulting in a layer of cells at the periphery of the
central vacuole.
|
Cytokinesis genes are required for endosperm cellularisation
To address the question of whether cellularisation and conventional
cytokinesis are under the same genetic and molecular control, we analysed
endosperm development in several cytokinesis mutants. Visual screens of
Arabidopsis embryos or seedlings with altered morphology have
identified numerous genes required for cytokinesis. Mutants with very similar
phenotypes include KNOLLE (KN)
(Mayer et al., 1991),
KEULE (KEU) (Mayer et
al., 1991
), HINKEL (HIN)
(Strompen et al., 2002
),
RUNKEL (RUK) (Nacry et
al., 2000
), OPEN HOUSE (OPN) (W. L. and C. S.,
unpublished) and PLEIADE (PLE)
(Hauser and Bauer, 2000
).
The phenotype of knolle and keule mutant embryos is easily discernible from the wild type, which has ordered files of regularly shaped cells (Fig. 2A). Both mutants are characterised by the presence of cells and nuclei of irregular shapes and sizes in addition to giant multinucleate cells (Fig. 2C,E). Nevertheless, the endosperm development is affected differently by these two mutations. In all keule seed analysed (n>30) cellularisation was indistinguishable from that in wild type, beginning at the MCE (Fig. 2C) and subsequently forming a layer of regularly positioned hexagonal cells in the PEN (Fig. 2D). Following cellularisation keule endosperm cells begin anticlinal division, forming a multilayered endosperm identical to that observed in wild-type seeds with early torpedo stage embryos. In contrast, knolle endosperm was not cellularised in the vast majority of seeds observed (n>50), with both MCE (Fig. 2E) and PEN (Fig. 2F) remaining syncytial. In approximately 10% of the seeds some degree of cellularisation was apparent, most frequently limited to the MCE. However, one case of cellularised PEN with multiple cell layers, containing multinucleated cells and irregularly shaped nuclei, was also observed. This is reminiscent of the phenotype variation observed in knolle embryos, containing a mixture of apparently normal and defective cells, with the difference that, being a synchronised event within each endosperm domain (MCE or PEN), defective cellularisation will be evident throughout the entire domain. Subsequently, individual cells might be affected by aberrant cytokinesis when additional cell layers are formed.
|
Double mutant knolle/keule embryos display a remarkable additive
phenotype, where cytokinesis is completely absent
(Fig. 2G)
(Waizenegger et al., 2000). In
accordance with the conclusion that KNOLLE is required for
cellularisation, the endosperm of knolle/keule seeds remained
syncytial (Fig. 2G,H). In
contrast to the embryo, no new endosperm phenotype was observed and the
endosperm phenotypes of knolle and keule/knolle mutants
appeared identical.
Endosperm cellularisation in plants mutant for HINKEL, which encodes a kinesin-related protein, and for OPEN HOUSE for which the gene product has not yet been identified, follows the pattern of knolle mutants. Although generally remaining syncytial (Fig. 3A-D), partial cellularisation, particularly in the MCE, was occasionally observed.
|
The endosperm phenotypes of runkel and pleiade are less
severe than those of knolle, hinkel and open house, although
cellularisation clearly requires these genes in order to proceed normally
(Fig. 3E-H). Partial
cellularisation of the MCE (Fig.
3E,G) was observed in almost all seeds analysed. Cellularisation
in the PEN, albeit defective and displaying multinucleate cells
(Fig. 3F), nuclei of different
sizes (Fig. 3H) and incomplete
cell walls (Fig. 3F,H) was
likewise observed in most seeds. In pleiade the relatively mild
cellularisation phenotype is accompanied by a correspondingly mild cytokinesis
phenotype, with the embryo maintaining a correct morphology until cotyledon
development (Fig. 3G).
Additionally, the first isolated mutant alleles of this gene allow germination
and plant development, with cytokinesis being affected only in the root
(Hauser and Bauer, 2000).
A common feature of the cellularisation mutants described here is the high density of NCDs aligned along the peripheral cell wall in the PEN (Fig. 2F,H, Fig. 3B,D). This indicates that, in the absence of cellularisation, the syncytial PEN will continue divisions at least through nine mitoses.
Periclinal cell wall formation is defective in
fass/ton2
The fass/ton2 mutation affects embryo morphogenesis by interfering
with cell wall orientation and cell shape, resulting in compressed embryos
with seemingly randomly arranged cells and abnormal tissue organisation
(Torres-Ruiz and Jürgens,
1994). Although endosperm cellularisation takes place in
fass/ton2 seeds, closer examination reveals differences between
wild-type and fass/ton2 endosperm. The number of cell layers in
completely cellularised seeds are frequently reduced from four in the wild
type (Fig. 4A) to two or three
in fass/ton2 (Fig.
4B). Observations at the cellularisation front, just after the
first anticlinal division reveal that these differences are caused by the
inability of the fass/ton2 mutant to correctly form periclinal cell
walls separating newly divided sister nuclei
(Fig. 4D). The layer of small
peripheral cells is not continuous in fass/ton2 endosperm and cells
of the inner layers of the PEN are longer than in the wild type possibly as a
result of the absence of periclinal divisions
(Fig. 4B,E).
Endosperm-specific cytokinesis-defective mutants define a novel gene,
SPÄTZLE
In an attempt to identify mutants that specifically affect endosperm
development but not embryonic development, we visually screened a subset of
150 lines from the Versailles collection of T-DNA insertion lines (20,000
lines pre-screened by L. L. for abnormal seed shape). Two lines, DRU42 and
DQB12, were recovered that showed phenotypes with aberrant endosperm
development but no apparent defect in the embryo. Complementation analyses
revealed the two mutations to be allelic. Upon selfing, 24.7% (s.e.m.=0.7;
n=500) of the seed from heterozygous plants displayed the mutant
endosperm phenotype, indicating that the mutations are recessive and segregate
in a mendelian fashion. In none of the lines was the mutant endosperm
phenotype linked with the kanamycin resistance gene of the T-DNA, leading to
the conclusion that the mutated gene is not tagged by a complete T-DNA.
Mapping with PCR based markers indicated that the SPÄTZLE gene
is located on chromosome 1 approximately 5 cM south of the SSLP marker
ACC2.
As with the other mutants that we analysed, spätzle displayed a variable range of phenotypes. At late-heart stage of embryogenesis, when endosperm cellularisation is complete in wild type (Fig. 2A), no sign of cell wall formation could be detected in the most frequent mutant phenotype, either in the MCE or in the PEN (Fig. 5). Until endosperm stage VIII, spätzle endosperm develops normally and the PEN contains regularly organised NCDs (Fig. 5C), as in wild type just prior to cellularisation. The PEN undergoes at least one additional mitotic division resulting in a syncytium with increased density of NCDs (Fig. 5D), similar to that of knolle, hinkel and open house as described above. Subsequently, the number of NCDs in the PEN continuously reduces while the size of individual nuclei increases. After stage VIII, we observed various degree of fusion of NCDs (Fig. 5E). Division of nuclei in these binucleate NCDs apparently leads to lack of nuclear separation upon mitosis (Fig. 5E) and ultimately to fusion of nuclei (Fig. 5F). This process can continue until the PEN contains a few giant NCDs, each with one or more giant multinucleolated nuclei (Fig. 5G). This pattern of endosperm development distinguishes spätzle from the other cytokinesis mutants where enlarged nuclei were always limited in frequency and accompanied by partial cellularisation of the PEN (e.g. Fig. 3H). At all stages analysed, in seeds displaying the spätzle endosperm phenotype embryos did not show any visible defects associated with cell division, such as incomplete cell walls, enlarged nuclei or multinucleate cells, which are typical of all the other cytokinesis mutants described here (Figs 2 and 3). Moreover, morphogenesis and pattern formation in spätzle embryos is normal (protoderm, vascular tissue, root and shoot embryonic meristems) (Fig. 5A). After torpedo stage, most spätzle embryos show retardation in overall growth (Fig. 5B), perhaps resulting from the inability of the defective endosperm to nurture the embryo. Mutant seeds, which can be recognised by their slightly shrunken appearance and slightly smaller size at maturity, are viable, can germinate on soil and give rise to homozygous spätzle plants. In those plants we could detect neither morphological defects nor growth retardation. Fertility was not altered, and homozygous spätzle plants produced 100% seeds with non-cellularised endosperm. Similar observations were made for both spätzle alleles, DRU42 and DQB12. These observations strongly support the conclusion that the SPÄTZLE gene product is required specifically for cellularisation in the endosperm but not for embryogenesis.
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Patterning of the endosperm is not dependent on cellularisation
We have previously shown that the FIS (Fertilisation
Independent Seed) genes are required for correct
anterior-posterior polarisation in the Arabidopsis endosperm and
that, additionally, endosperm cellularisation is impaired in a fis
mutant background (Sørensen et al.,
2001). To investigate whether a general connection exists between
cellularisation and patterning, we analysed development of the CZE in two
representative cytokinesis mutants. As in wild type
(Fig. 6A), knolle
posterior pole endosperm is characterised by the presence of a chalazal cyst
and one or more nodules, both containing multiple nuclei in a common cytoplasm
(Fig. 6C). Likewise,
spätzle CZE morphology appears normal
(Fig. 6E), but the cytological
distinction between chalazal nodules and giant NCDs in the PEN is not as clear
as in the wild type or as in knolle. In the two mutants
knolle and spätzle we introgressed the marker line
(KS117) with an exclusive GFP expression at the posterior pole of the
endosperm (Fig. 6B). The
expression of the marker is restricted to the CZE in all seeds of both
knolle (Fig. 6D) and
spätzle (Fig. 6F)
siliques. This confirms the correct patterning of the endosperm in the absence
of cellularisation in cytokinesis-defective mutants as well as in
spätzle. Cellularisation therefore does not appear to be
necessary for the expression of endosperm polarity.
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DISCUSSION |
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Among the genes investigated, KNOLLE, KEULE and HINKEL
have been cloned and found to encode proteins potentially involved in vesicle
trafficking. HINKEL encodes a novel kinesin that is localised to the
cell plate during cytokinesis in a pattern complementary to that of the KNOLLE
protein (Strompen et al.,
2002). KNOLLE is localised to forming cell plates in cellularising
endosperm (Lauber et al.,
1997
), in agreement with the absence of cellularisation observed
in knolle. Direct interaction between the KNOLLE syntaxin and the
KEULE sec1-related protein has been demonstrated and it is therefore
surprising that keule mutant endosperm has a wild-type appearance. It
is not known whether KEULE is present in the endosperm. In contrast to the
embryo, the endosperm of the knolle keule double mutant does not show
an additive phenotype to that observed in knolle. It is thus likely
that KEULE either is not expressed during endosperm cellularisation or that a
functionally redundant gene exists. It has been suggested that each Sec1
protein interacts with several syntaxins
(Sanderfoot et al., 2000
). Our
observation that KNOLLE but not KEULE is required for endosperm
cellularisation suggests that the KNOLLE syntaxin is likely to interact with
other Sec1 proteins than KEULE in the endosperm.
Absence of endosperm cellularisation has also been reported in mutants
affected in structural elements such as microtubules. Mutations in the
PILZ group of genes (Mayer et
al., 1999) that includes ttn1 and ttn5
(McElver et al., 2000
;
Tzafrir et al., 2002
) affect
the complex of tubulin-folding cofactors and cause disruption to the
microtubule cytoskeleton (Steinborn et
al., 2002
). The absence of microtubules thus explains the lack of
cytokinesis in the embryo and of cellularisation in the endosperm of
pilz mutants. Other titan mutants
(Liu and Meinke, 1998
),
including titan-7 and -8 encoding chromosome scaffold
proteins (Liu et al., 2002
)
show a non-cellularised endosperm with giant nuclei likely to be the indirect
consequence of defective chromosome separation.
Hitherto, comparisons of cellularisation with conventional cytokinesis has
been based on different cytological analyses of wild-type tissues, and
although the similarities of the two processes has been acknowledged
(Olsen, 2001) most reports
have emphasised their differences (Otegui
and Staehelin, 2000a
; Nacry et
al., 2000
; Verma,
2001
). Our genetic analysis of the knolle, hinkel, open house,
runkel and pleiade mutations directly involved in cytokinesis
provides conclusive evidence that somatic cytokinesis and endosperm
cellularisation share many components.
FASS is not required for endosperm cellularisation
The PPB is a temporary assemblage of microtubules that marks the future
site of connection of the cell plate to the cell wall in plant cytokinesis and
results in an F-actin-free zone. PPBs have not been observed during endosperm
cellularisation but only during the subsequent developmental phase of the
barley aleurone layer (Brown et al.,
1994). In Arabidopsis, both cellularisation and
subsequent periclinal divisions take place without a discernible PPB
(Brown et al., 1999
). The PPB
is lacking in fass/ton2 mutants and interestingly, fass/ton2
cellularisation occurs normally in MCE and PEN but subsequent cytokinesis is
impaired in the endosperm. This confirms that a PPB is not required for
cellularisation, but, in contrast to earlier observations, it appears to
indicate that the PPB is necessary for the first periclinal divisions to
proceed correctly. Alternatively, the FASS gene product might be
involved in an unknown process or structure directing periclinal divisions in
Arabidopsis endosperm.
Endosperm cellularisation is coupled to mitosis as conventional
cytokinesis
In previous observations on fixed seeds cellularisation appeared to
progress as a wave from the MCE towards the CZE
(Mansfield and Briarty,
1990b). Cellularisation was considered to be uncoupled from
mitosis and no distinction was seen in the appearance of cell walls between
sister or non-sister nuclei (Brown et al.,
1999
). In order to perform real time in vivo observations of this
process we used the GFP variant mGFP5 that is targeted to the endoplasmic
reticulum and accumulates around the forming cell plate in root meristem cells
(Haseloff, 1999
). Accumulation
of RE on the side of cell plates in endosperm has been reported
(Otegui et al., 2001
) and this
likely accounts for the labelling with mGFP5 that we observed. This property
allowed us to visualise the time course of cellularisation in living seeds.
Completely cellularised MCE was observed at the end of the globular stage of
embryogenesis. At this stage, the endosperm contains approx. 100 nuclei (stage
VIII). The wave of mitosis leading to stage IX is initiated in the MCE and
propagates within 1 hour through the PEN, as observed already during syncytial
divisions (Boisnard-Lorig et al.,
2001
). In vivo observations enabled us to establish that
cellularisation in the PEN is taking place in conjunction with the 8th mitotic
cycle. The cell walls first appear between sister nuclei, rapidly followed by
walls completely surrounding individual NCDs, thereby defining cells. Although
the connection between mitosis and cellularisation has not previously been
observed in Arabidopsis, probably due to the short time scale (one
hour) of these events, coincidence of cell plate initiation with nuclear
division has been remarked on in Ranunculus sceleratus
(Xu and van Lammeren, 1993
).
Indeed we observed typical intermediate structures with nascent cell walls
forming between sister nuclei only in three fixed seeds amongst several
thousands. Furthermore, our observations relate to the initial appearance of
walls, not to the rate at which these might expand anticlinally to form the
characteristic open-ended alveoli (Brown et
al., 1999
).
In Arabidopsis endosperm the link between cellularisation and
mitosis reported in this study furthers the parallels between endosperm
development and Drosophila embryogenesis. In Drosophila a
series of rapid succession of pseudo-synchronous waves of mitosis cross the
embryo until cycle 10. Then a marked and gradual increase in the length of the
cell cycle culminates at cycle 14 in the cellularisation of the blastoderm
(Edgar et al., 1986). In
Drosophila, the signal that initiates blastoderm cellularisation
relies on a nucleo-cytoplasmic threshold
(Edgar et al., 1986
). This
might be the case as well in the Arabidopsis endosperm.
The spätzle mutant phenotype points to the existence of
a cellularisation-specific checkpoint
We have isolated two alleles of a new mutant, spätzle, that
are characterised by the absence of cellularisation in the endosperm but do
not show cytokinesis defects in the embryo. Mature seeds will germinate and
the homozygous spätzle plant is indistinguishable from wild
type. One possible explanation for the absence of defects in embryos and
homozygous vegetative tissues is that SPÄTZLE encodes a gene
involved in mechanisms common to cellularisation and cytokinesis, but
functional redundancy prevents the appearance of an embryo phenotype, a
situation that could mirror our interpretation of defects observed in
keule embryos but not in the keule endosperm (see discussion
above). Alternatively, spätzle mutants might be affected in a
process specific to cellularisation.
Much like spätzle, the fis mutants produce endosperm
that does not cellularise although the embryo does not display any defect in
cytokinesis, is viable in fis1/medea and in fis2 mutants
(Sørensen et al., 2001)
and can produce plants of wild-type appearance. The FIS genes,
control multiple aspects of seed development including initiation of endosperm
divisions (Ohad et al., 1999
;
Luo et al., 1999
) and correct
definition of the CZE (Sørensen et
al., 2001
). In contrast, endosperm polarity is clearly not
affected in spätzle endosperm and the SPÄTZLE and
FIS genes are probably not in the same genetic pathway.
Like spätzle, titan3 does not affect embryo development, with
the mutant phenotype being restricted to the endosperm, where giant nuclei and
NCDs are formed (Liu and Meinke,
1998). However, ttn3 endosperm is capable of
cellularisation that takes place at the same time as in the wild-type seeds.
The defective cellularisation in spätzle endosperm is probably
not a consequence of nuclei enlargement. The gene product of
SPÄTZLE is indeed required for and limited to the process of
cellularisation, possibly being a structural protein specifically expressed in
the endosperm. As an alternative hypothesis we propose that the primary defect
in spätzle is related to the nucleo-cytoplasmic ratio or to its
interpretation. Cloning the gene mutated in spätzle might allow
distinction between these hypotheses.
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ACKNOWLEDGMENTS |
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Footnotes |
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