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First published online 4 October 2006
doi: 10.1242/dev.02599
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1 Institute of Plant Sciences and Basel-Zurich Plant Science Center, ETH Zurich,
8092 Zurich, Switzerland.
2 Department of Plant and Microbial Biology, University of California, Berkeley,
CA 94720, USA.
* Author for correspondence (e-mail: lars.hennig{at}ipw.biol.ethz.ch)
Accepted 30 August 2006
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SUMMARY |
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 TOP SUMMARY INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Key words: Arabidopsis, CAF-1, Chromatin, Endoreduplication, MSI1, Shoot apical meristem, Trichome
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INTRODUCTION |
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TOPSUMMARYINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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; Mello and Almouzni,
2001; Loyola and Almouzni,
2004). This heterotrimeric complex is found in yeast, animals and
plants, and facilitates nucleosome assembly on newly replicated DNA
(Smith and Stillman, 1989;
Bulger et al., 1995;
Verreault et al., 1996;
Kaufman et al., 1997;
Kaya et al., 2001). Human CAF1
binds newly synthesized H3-H4 histone dimers and, because its largest subunit
(p150) physically interacts with the proliferating cell nuclear antigen PCNA,
these histones are targeted to regions of DNA synthesis during replication or
repair (Gaillard et al., 1996;
Verreault et al., 1996;
Shibahara and Stillman, 1999;
Moggs et al., 2000;
Zhang et al., 2000;
Krawitz et al., 2002).
Importantly, all three CAF-1 subunits physically interact with other proteins
as well: the smallest subunit, p48, is found in multiple complexes affecting
chromatin dynamics (for a review, see
Hennig et al., 2005); the
second largest subunit, p60, interacts with the histone chaperone
anti-silencing factor 1 (Mello et al.,
2002); and p150 interacts with heterochromatin protein 1,
methyl-binding protein 1 and the Bloom's syndrome DNA-helicase BLM
(Murzina et al., 1999;
Reese et al., 2003;
Jiao et al., 2004;
Sarraf and Stancheva, 2004).
However, it is unclear whether CAF-1 formation and the other interactions
described above occur simultaneously, or whether they are exclusive,
indicating CAF-1-independent functions of the subunits.
Yeast mutants deficient in CAF-1 subunits are viable but show enhanced
sensitivity to UV light (Kaufman et al.,
1997; Game et al., 1999). In addition, maintenance of silencing at
mating type loci and near the telomeres is impaired
(Enomoto et al., 1997;
Kaufman et al., 1997;
Monson et al., 1997;
Enomoto and Berman, 1998;
Smith et al., 1999;
Taddei et al., 1999). Unlike
in yeast, much less is known about the role of CAF-1 in the life cycle of
multicellular organisms, mainly because mutants are not readily available.
Transgenic approaches were therefore used to analyze CAF-1 function during the
development of model organisms. For example, expression of a truncated version
of human p150 interfered with the maintenance of transcriptional gene
silencing in mammalian cells (Tchenio et
al., 2001), and expression of a truncated version of p150 in
Xenopus caused severe defects in embryo development, suggesting that
CAF-1 is essential in vertebrates (Quivy
et al., 2001). This hypothesis was strongly supported by RNAi
knock-down experiments of p150 in human cell lines. Such cells showed delayed
DNA replication and accumulated in S-phase
(Hoek and Stillman, 2003).
Similarly, silencing of p60 expression by RNAi caused apoptosis in dividing,
but not in quiescent, human cells
(Nabatiyan and Krude,
2004).
The model plant Arabidopsis thaliana is currently the only
multicellular organism for which mutants in all three CAF-1 subunits are
available. Reconstitution experiments showed that FASCIATA 1 (FAS1, a homolog
of p150), FAS2 (a homolog of p60) and Arabidopsis MSI1 (a homolog of
p48) form a functional CAF-1 complex in vitro
(Kaya et al., 2001). Mutants
deficient in FAS1 and FAS2 were originally identified because of their
fasciated phenotype (Reinholz,
1966; Leyser and Furner,
1992). Fasciation describes a set of developmental abnormalities
caused by defects of the shoot apical meristem (SAM) that includes altered
phyllotaxis, broadening and bifurcation of the stem, and alterations in the
number of floral organs (Worsdell,
1905). The SAM contains slowly dividing stem cells that give rise
to all cells in above-ground, post-embryonic plant organs
(Scheres et al., 2004). Proper
organization of the SAM depends on several proteins, including the key
regulator WUSCHEL (Laux et al.,
1996). The fasciated phenotype of fas1 and fas2
is caused at least in part by loss of the restricted spatial expression
pattern of WUSCHEL (Kaya et al.,
2001). In addition to SAM defects, fas1 and fas2
have defects in the root apical meristem (RAM), which produces the cells
needed for post-embryonic root growth
(Scheres et al., 2004). This
is because the restricted spatial expression patterns of the RAM key regulator
SCARECROW are partially lost in the fasciata mutants
(Kaya et al., 2001).
Surprisingly, plants lacking both FAS1 and FAS2 subunits of CAF-1 are viable
and fertile, but express some silenced transposons or transgenes
(Takeda et al., 2004;
Ono et al., 2006). By
contrast, msi1-1 null mutants are not viable and seeds abort early
during embryo development (Köhler et
al., 2003; Guitton et al.,
2004). MSI1 co-suppression (msi1-cs) plants with strongly
reduced protein levels (>90% reduction) are viable but show severe
developmental defects, including homeotic transformation of floral organs and
sterility (Hennig et al.,
2003). Because such defects were not observed in the
fasciata mutants, it was concluded that they are caused by the
reduced function of other MSI1-containing complexes, such as the MEDEA (MEA)
or CURLY LEAF Polycomb group complex
(Hennig et al., 2003;
Köhler et al., 2003;
Hennig et al., 2005;
Schönrock et al.,
2006a).
Thus, CAF-1 is required for the execution of normal developmental programs in the apical meristems, but roles for CAF-1 in other tissues are much less understood. Here, we show that expression of FAS1 and FAS2 is high in actively dividing cells, and that several aspects of development, including seedling growth and leaf hair differentiation, are impaired in Arabidopsis mutants lacking CAF-1. Therefore, CAF-1 plays a more general role in plant development than has been previously appreciated.
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MATERIALS AND METHODS |
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TOPSUMMARYINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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; Kaya et al.,
2001), fas2-1 (Leyser
and Furner, 1992; Kaya et al.,
2001) and gl3-1
(Koornneef et al., 1982;
Payne et al., 2000) mutants
were obtained from the Nottingham Arabidopsis Stock Centre. New fas1
and fas2 alleles were identified in T-DNA insertion mutant
collections (Sessions et al.,
2002; Alonso et al.,
2003), and named fas1-4 and fas2-4 (see Figs S1,
S2 in the supplementary material). Plants were kept in Conviron growth
chambers with mixed cold fluorescent and incandescent light (110 to 140
µmol/m2s, 21±2°C) under long-day (LD, 16 hour light)
photoperiods, unless indicated otherwise. For characterization of seedling
development, seeds were plated on four layers of water-soaked filter paper,
which were placed into clear plastic boxes. A 48-hour dark treatment at
4°C was followed by induction of germination by white light for 10 hours
and further incubation of seedlings in the dark at 23°C or under LD
photoperiods.
Generation of transgenic plants with reduced MSI1 protein levels
In contrast to the viable fas1-1 and fas2-1 mutants,
which have premature stop codons and are most likely null alleles
(Kaya et al., 2001), loss of
MSI1 is lethal (Köhler et al.,
2003; Guitton et al.,
2004). Therefore, we constructed transgenic lines in which the
level of MSI1 was only moderately decreased by a MSI1 antisense
construct. A 532-bp ClaI/XbaI fragment of the MSI1
coding sequence was fused in antisense orientation to the cauliflower mosaic
virus (CaMV) 35S promoter in the binary vector pSLK7292 and transformed into
Col wild-type plants. We selected the line 1ASb7 (msi1-as), which
segregated a single transgene in a 1:3 ratio and had 30-50% of wild-type MSI1
protein levels (see Fig. S3A in the supplementary material), for further
characterization, but the phenotypes described below were observed for several
independent lines. Similar to fas1-1 and fas2-1, but in
contrast to msi1-cs and msi1-1, msi1-as plants were fertile
and showed no seed abortion. Development of msi1-as plants was
delayed, and plants had a reduced rosette size, a delayed flowering time and
reduced growth of primary shoots, although more severe symptoms of fasciation,
such as stem bifurcation, were not observed (see Fig. S3C in the supplementary
material; data not shown). MSI1 shares only 25-52% identity with the other
MSI1-like proteins in Arabidopsis, suggesting that expression of the
MSI1 antisense-RNA construct would not affect expression of
MSI2-5; and semi-quantitative RT-PCR did not reveal any changes in
MSI2-5 transcript levels in msi1-as plants (see Fig. S3B in
the supplementary material). In addition, we could not detect elevated
transcript levels for AGAMOUS (AG) or APETALA2
(AP2) in msi1-as leaves (see Fig. S3D in the supplementary
material). AG and AP2 are floral homeotic genes that are
ectopically expressed in non-floral tissues of msi1-cs plants, but
not in fas1-1 or fas2-1 mutants
(Hennig et al., 2003).
Protein gel blot analysis
Protein extracts and protein gel blots using a specific anti-MSI1 antiserum
were performed as described previously (Ach
et al., 1997; Hennig et al.,
2003).
RNA isolation and RT-PCR
RNA was extracted as previously described
(Hennig et al., 2003). For
RT-PCR analysis, 0.4-1.0 µg total RNA was treated with DNase I. The
DNA-free RNA (0.2-1.0 µg) was reverse-transcribed using an oligodT primer
and MMLV reverse transcriptase (Clonetech, Palo Alto, CA). Aliquots of the
generated cDNA were used as template for PCR with gene-specific primers
(Table 1).
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Ploidy analysis
Tissue was cut into small pieces in 400 µl nuclear extraction buffer
(Partec, Münster, Germany), incubated for 30 minutes on ice, filtered
through a 30 µm mesh, mixed with 1 ml nuclear staining buffer (Partec) and,
after further incubation on ice for 10 minutes, analyzed with a Partec Ploidy
Analyzer. For quantification, the results of three to six independent
preparations were averaged.
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).
Briefly, young rosette leaves were fixed in FAA (50% ethanol, 5% glacial
acetic, 10% formaldehyde). Staining was performed for 1.5 hours with 130
µg/ml DAPI in McIlvaines buffer (60 mM citric acid, 80 mM sodium phosphate,
pH 4.1). Samples were washed once for 15 minutes and once for 1 hour with
McIlvaines buffer, and mounted in McIlvaines buffer with 50% glycerol.
Fluorescence was recorded with a MagnaFire CCD camera (Optronics, Goleta, CA),
or with an Apogee Alta U32 CCD camera (Apogee Instruments, Roseville, CA).
Images were quantified using ImageJ. Total fluorescence of at least 30
representative nuclei per experiment was determined and calibrated using guard
cell nuclei (n
30), which are considered to be strictly diploid
(Walker et al., 2000).
Analysis of leaf histology
Plants were grown until bolting before leaves were harvested. Leaf area was
determined by scanning the leaves and measuring their size with ImageJ. Leaves
were then fixed in ethanol:acetic acid (9:1) at room temperature for 2.5
hours, dehydrated in 90% then 70% ethanol for 1 hour each, and cleared in
clearing solution [66.7% (w/v) chloral hydrate, 8.3% (w/v) glycerol in water]
at 4°C overnight. Samples were mounted in clearing solution and
investigated with DIC optics. Images were recorded with an AxioCam HRc CCD
camera and analyzed with ImageJ.
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RESULTS |
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TOPSUMMARYINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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). Accordingly, the expression of the two large
CAF-1 subunits increases in proliferating Arabidopsis cells, and is
cell cycle regulated in Arabidopsis and yeast
(Kaya et al., 2001) (see Fig.
S4 in the supplementary material). By contrast, expression of the small
subunit MSI1 does not strongly correlate with cell proliferation or cell
cycle. These expression patterns reflect the more widespread role of MSI1 in
other chromatin complexes (Hennig et al.,
2005). The expression data suggest that CAF-1 is required in
dividing cells. However, to date, only defects in meristem function have been
studied in detail for fasciata mutants
(Reinholz, 1966;
Leyser and Furner, 1992;
Kaya et al., 2001). To test
whether CAF-1 function is required in other organs as well, we characterized
meristem-independent cell proliferation and differentiation in CAF-1 mutants.
fas1 and fas2 alleles have been previously characterized in
Landsberg erecta, Enkheim or Nossen
(Reinholz, 1966; Leyser et
al., 1992; Kaya et al., 2001).
To test whether any defects observed are genuine traits of CAF-1 loss or are
caused by the genetic background of the different accessions, we identified
novel fas1 and fas2 alleles in Columbia - fas1-4
and fas2-4 (see Figs S1, S2 in the supplementary material). The
fas1-4 and fas2-4 phenotypes are similar to the phenotypes
of other fasciata mutants: the plants are smaller than wild type and
develop fasciation. Seedling organs such as hypocotyl and cotyledons are not formed by the apical meristems, but are formed during embryogenesis. Therefore, we analyzed seedlings of CAF-1 mutants in more detail. General seedling morphology was not severely affected in fas1, fas2 or msi1-as seedlings grown in long-day photoperiods (Fig. 1A, see also Figs S1, S2 in the supplementary material), but msi1-as seedlings consistently showed cotyledon epinasty, i.e. a downward curling of the cotyledons. As this phenotype was not observed in fas1 or fas2 seedlings, but was sometimes apparent in msi1-cs lines (P. Taranto, PhD thesis, University of California, Berkeley, 1998), we conclude that it is not related to CAF-1 but rather to a different MSI1-function. Dark-grown seedlings of all three CAF-1 mutants had shorter hypocotyls than did wild type (Fig. 1B); this phenotype was most pronounced for fas1-1, and weakest for fas1-4 and msi1-as. Note that some FAS1 transcript is made in the fas1-4 insertion mutant (see Fig. S1 in the supplementary material), demonstrating that fas1-1 but not fas1-4 is a null allele. By contrast, both fas2 alleles appear to be null (see Fig. S2 in the supplementary material). When seedlings were grown under continuous white light, all plants responded strongly with inhibition of hypocotyl elongation (data not shown). The short hypocotyls formed in the light precluded the detection of length differences between CAF-1 mutants and wild type.
Post-germination hypocotyl growth in the dark results mostly from cell
elongation and only a few cell divisions occur
(Gendreau et al., 1997). To
determine whether the shorter hypocotyls in dark-grown CAF-1 mutant seedlings
are caused by defects in cell division or elongation, we counted hypocotyl
epidermal cell number and measured cell size
(Fig. 2A,B). The results show
that hypocotyl epidermal cells of both fas2 alleles were
significantly shorter than in the wild-type, whereas cell size was not, or
only weakly, affected in fas1-1, fas1-4 and msi1-as. These
results suggest that the reduced size of hypocotyl epidermal cells is a
genuine trait of fas2 mutants and is not caused by the genetic
background. Moreover, loss of CAF-1 does not necessarily cause size reductions
for hypocotyl epidermal cells. By contrast, seedlings lacking any of the three
CAF-1 subunits had reduced cell numbers in their hypocotyls. This reduction
was strongest in fas1-1 and weakest in msi1-as. Only the
weak fas1-4 allele had slightly increased cell numbers.
Interestingly, hypocotyls of fas1, fas2 and msi1-as
seedlings were significantly thicker than hypocotyls of wild-type plants
(Fig. 2C). Notably, the
increase in hypocotyl diameter was strongest in the weak fas1-4
allele, whereas changes in cell size and cell number were smallest in this
allele. In summary, CAF-1 subunits are therefore not only required to control
cell proliferation in the SAM and RAM, but also for normal cell proliferation
in hypocotyls.
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), and cell elongation and endoreduplication are often
tightly coupled. Because cell elongation is impaired in fas2
hypocotyls, we measured DNA endoreduplication in dark- and light-grown CAF-1
mutant seedlings. However, rather than a decrease in endoreduplication,
seedlings grown in long-day photoperiods or in the dark showed increased DNA
endoreduplication (Fig. 3A,B):
the fraction of 2C nuclei decreased from 30-40% to 20-30%, whereas the
fraction of 8C and 16C nuclei increased. This shift in ploidy was strongest
for fas1-1, fas1-4 and fas2-4, but was less pronounced for
msi1-as. We also measured ploidy directly in nuclei of
fas1-1 and wild-type hypocotyl cells, and found that the increased
DNA content was caused, at least partially, by higher ploidy in the hypocotyl
(data not shown). Interestingly, ploidy patterns in fas2-1 did not
differ from those of wild-type Landsberg erecta. Thus, CAF-1
deficiency does not generally affect DNA endoreduplication, and the reduced
cell size in dark-grown fas2-1 and fas2-4 hypocotyls does
not correlate with reduced endoreduplication.
Size of leaf epidermal cells is altered in CAF-1 mutants
The SAM produces leaf primordia, but the final leaf morphology is mostly
independent of SAM function (Byrne,
2005; Fleming,
2005). The smaller rosette diameter and the serration of rosette
leaves in fasciata mutants in the Col and En accessions
(Reinholz, 1966;
Leyser and Furner, 1992;
Serrano-Cartagena et al.,
1999; Kaya et al.,
2001) suggested that cell proliferation and/or expansion was
affected during leaf development. To test this hypothesis, the histology of
rosette leaves was investigated. In addition to the fas1-1 allele in
En, we included the novel fas1-4 allele in the Col background. The
phenotype of fas1-4 plants was similar to that of other
fasciata mutants, including alterations in phyllotaxis and reduced
shoot height (see Fig. S1 in the supplementary material). Furthermore,
fas1-4 rosette leaves were serrated, similar to the rosette leaves of
fas1-1 and fas2-4 plants. Growth of rosette leaves occurs in
two phases: outgrowth through cell division and enlargement of the blade
through cell expansion (Beemster et al.,
2005). Cross sections through the first or second rosette leaves
of plants at bolting did not reveal any obvious differences in internal leaf
histology between wild type and mutants (data not shown). By contrast,
epidermal cells of these leaves were significantly larger in the CAF-1 mutants
than in the wild type (Fig.
4A,B). This effect was much stronger in the fas1-4 and
fas2-4 mutants (1.5 to 2-fold increase) than in the msi1-as
plants (1.2-fold increase).
Next, we determined the number of leaf epidermal cells. Consistent with the observation that CAF-1 mutations interfere with regulation of cell division rates during hypocotyl development, the number of epidermal cells was reduced in rosette leaves of both fas1-4 and fas2-4 (Fig. 4C). By contrast, the number of epidermal cells was not significantly altered in msi1-as plants. Similar results were obtained when the third and fourth rosette leaves were analyzed (Fig. 4D,E). These results show that CAF-1 is required not only to control cell proliferation in the SAM, RAM and hypocotyls, but also for normal cell proliferation in expanding rosette leaves. In addition, these results suggest that the phenotype of fasciata mutants does not become more severe during plant development.
Trichome differentiation is altered in CAF-1 mutants
Trichomes are leaf hairs that originate from the leaf or stem epidermis. In
Arabidopsis, each trichome consists of a single cell and therefore
provides an ideal system with which to study cell differentiation. Lateral
inhibition maintains a regular spacing of trichomes in wild-type leaves. After
trichome fate commitment, cells stop dividing but continue to synthesize DNA.
The differentiating trichome cell extends out of the leaf surface, undergoes
two branching events and finally elongates extensively
(Schnittger and Hülskamp,
2002; Larkin et al.,
2003). The majority of trichomes on Arabidopsis leaves
have three branches (Fig. 5A).
By contrast, CAF-1 mutants contain many trichomes that develop more than three
branches (Fig. 5B,G). In
general, only about 80% of trichomes in CAF-1 mutants have three branches, but
up to 30% have four to six branches (Fig.
5G). Similar results were obtained when the third and fourth
leaves were analyzed (data not shown), suggesting that this trichome phenotype
is stable throughout development. The leaves of CAF-1 mutants did not contain
clustered trichomes suggesting that regular trichome initiation does not
require CAF-1.
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|
). Microscopic investigation of DAPI-stained trichomes
revealed that trichomes of CAF-1 mutants are also single cells with one
nucleus (Fig. 5C-F). During
trichome differentiation, four rounds of DNA endoreduplication usually occur,
which establish a relative DNA content of 32C instead of the usual 2C of
diploid cells. Because increased trichome branching is often correlated with
extra DNA endoreduplication, we measured the DNA content in trichomes of CAF-1
mutants (Fig. 5H). Only
trichome nuclei of fas1-1 and fas1-4 consistently contained
more DNA in all experiments, suggesting that many trichome cells undergo at
least one extra round of DNA endoreduplication. Although fas2-4 had a
non-significant tendency for increased ploidy, this was not evident in all
experiments, despite consistently increased trichome branching. Because
fas2-1, fas2-4 and msi1-as trichomes develop extra branches
without significantly increased ploidy levels, we reasoned that CAF-1 controls
trichome branching via a pathway that is independent of DNA
endoreduplication.
FAS1, FAS2 and MSI1 act together in trichome development
Although the similar phenotypes of fas1, fas2 and msi1-as
plants strongly suggest that FAS1, FAS2 and MSI1 function in CAF-1 during
trichome development, the different effects on ploidy make it possible that
they affect trichome development independently of CAF-1. To test this
possibility genetically, we generated fas1-4 fas2-4, fas1-4 msi1-as
and fas2-4 msi1-as double mutants. All of these double mutants were
viable, similar to the previously described fas1-1 fas2-1 double
mutant (Hennig et al., 2003),
and no obvious synergistic effects were observed during development. In
particular, quantification of trichome branching showed no additive or
synergistic effects between fas1-4, fas2-4 and msi1-as
(Fig. 6A). Similarly,
fas1-1 fas2-1, fas1-1 msi1-as and fas2-1 msi1-as double
mutants generated by inter-accession crossings did not show increased trichome
branching (data not shown). These results demonstrate that FAS1, FAS2
and MSI1 function in the same genetic pathway to control trichome
development, possibly by acting together in the CAF-1 complex.
|
),
and gl3-1 mutants produce trichomes with less than three branches
(Payne et al., 2000). To
genetically test the hypothesis that CAF-1 controls trichome development in a
pathway parallel to the endoreduplication-dependent pathway, in which
GL3 acts, we generated fas2-1 gl3-1 double mutants. Whereas
fas2-1 had almost no single-ended but many three-ended trichomes,
gl3-1 had 24% single-ended and only 2% three-ended trichomes. By
contrast, the trichome branching phenotype of the fas2-1 gl3-1 double
mutant was intermediate to the two single mutants, with only 4% single-ended
trichomes and 10% three-ended trichomes
(Fig. 6B). Because
FAS2 and GL3 had an additive rather than epistatic
interaction, CAF-1 acts in a pathway genetically parallel to the
GL3-containing endoreduplication pathway. |
DISCUSSION |
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TOPSUMMARYINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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;
Leyser and Furner, 1992;
Kaya et al., 2001). In
particular, defects in the organization of the SAM were suggested to cause
abnormal phyllotaxis and a thickening or even bifurcation of the stem. These
defects, together with the reduced growth rate of the primary shoot, are most
likely the consequence of disruptions in normal SAM function. However, the
fasciata mutants also have altered leaf shapes
(Reinholz, 1966;
Leyser and Furner, 1992).
Because leaf shape is mainly established during outgrowth of the leaf
primordia (Fleming, 2002;
Tsukaya, 2003), these
observations suggest that CAF-1 function is also required in organ primordia
and developing lateral organs. This conclusion is supported by the strong
expression of FAS1 in leaf primordia
(Kaya et al., 2001). In
contrast to the post-embryonic organs of a mature plant, hypocotyl and
cotyledons are formed during embryogenesis independently of the SAM. During
initial seedling development, very little cell division occurs, and this is
restricted mainly to the cotyledons
(Gendreau et al., 1997).
Growth is controlled by cell elongation, which is often paralleled by DNA
endoreduplication. We found that CAF-1 mutant seedlings have shorter
hypocotyls in the dark and that this is mainly due to reduced cell number
rather than altered cell size. Therefore, CAF-1 deficiency impairs normal
development by interfering with cell proliferation as early as during
embryogenesis or seedling development.
|
). This could
explain why, despite the greatly reduced numbers of epidermal cells, the leaf
area is often only mildly reduced in fas1-4 and fas2-4
mutants (data not shown). Furthermore, altered cell number and cell size could
potentially account for the change in trichome numbers on rosette leaves of
CAF-1 mutants, because it was primarily the trichome density relative to leaf
size, rather than the trichome density relative to epidermal cell number, that
was reduced (data not shown). Thus, CAF-1 is needed for efficient cell
proliferation in developing hypocotyls and rosette leaves, and compensation by
altered cell size might occur preferentially in leaves but not in
hypocotyls.
CAF-1 is required for trichome differentiation
Trichome morphogenesis is a well-studied developmental process, and many
mutants with altered trichomes have been identified
(Oppenheimer, 1998;
Schnittger and Hülskamp,
2002; Larkin et al.,
2003). The genetic analysis of branching mutants suggests that
several redundant pathways control branch formation
(Luo and Oppenheimer, 1999).
One group of genes affecting trichome branching primarily controls the number
of DNA endoreduplication cycles, which in turn determines branch number
(Hülskamp et al., 1994;
Perazza et al., 1999;
Kirik et al., 2001).
GL3 is one member of this group and encodes a bHLH protein that
positively regulates endoreduplication and branching events in trichomes
(Payne et al., 2000). A second
group of branching mutants affects branch number independently of DNA
endoreduplication (Folkers et al.,
1997; Luo and Oppenheimer,
1999; Qiu et al.,
2002). In all CAF-1 mutants, trichome patterning was maintained
and no trichome clusters were observed, indicating that CAF-1 is not required
for commitment to the trichome cell fate. Similarly, trichomes in fas1,
fas2 and msi1-as, and in the double mutants we investigated,
were single cells containing one nucleus, suggesting that most steps in
trichome differentiation are completed normally even in the absence of CAF-1.
However, all CAF-1 mutants had increased branch numbers. Interestingly,
increased trichome branching was also observed in Hosoba toge toge, a
deletion mutant lacking a 75.8-kb region encompassing 15 genes, including
FAS1 (Kaya et al.,
2000). Because only fas1 trichomes had a consistently
increased DNA content, the increased branching is not strictly correlated with
DNA endoreduplication. Therefore, CAF-1 functions either in a DNA
endoreduplication-independent pathway or downstream of DNA endoreduplication
to control trichome branching. This hypothesis is also supported by the fact
that the gl3-1 fas2-1 double mutants analysed have intermediate
branch numbers and none of the two alleles was epistatic over the other. Most
likely, CAF-1 functions in a DNA endoreduplication-independent pathway for
trichome branching. In summary, CAF-1 is not generally needed for cell fate
determination, but it is needed for normal cell proliferation and, in
trichomes, even for proper differentiation.
CAF-1-independent functions of CAF-1 subunits
Mutants lacking any one of the three subunits of Arabidopsis CAF-1
display a range of similar phenotypes that is consistent with the loss of
CAF-1 activity. Similarly, CAC1, CAC2 and CAC3 are all required for CAF-1
function in yeast (Game and Kaufman,
1999). However, some phenotypes caused by reduced FAS1, FAS2 or
MSI1 levels differ, suggesting that CAF-1 subunits can function independently
of CAF-1. For instance, only the loss of FAS2 led to smaller
hypocotyl epidermal cells independent of the genetic background. Likewise,
msi1-as plants show unique features, in particular they often develop
cotyledon epinasty. Because this trait was never observed in fas1 or
fas2, it most likely resulted from the lack of a CAF-1-independent
MSI1 function. In addition, the previously described msi1-cs plants
develop severe phenotypic defects, including homeotic changes of floral organ
identity, which are not present in fas1, fas2 and msi1-as
(Hennig et al., 2003). Embryos
of the msi1-1 mutant abort early in development, but embryos of
fas1, fas2 and msi1-as show no obvious developmental defects
(Kaya et al., 2001;
Köhler et al., 2003;
Guitton et al., 2004).
Biochemical analyses suggest that MSI1-like proteins participate in several
protein complexes acting on chromatin, including histone deacetylases,
chromatin remodelling factors and Polycomb Group protein complexes (for a
review, see Hennig et al.,
2005). We found that Arabidopsis MSI1, in addition to
FAS1, also interacts with fertilization-independent endosperm (FIE), histone
deacetylase, and the retinoblastoma-related RBR protein
(Ach et al., 1997;
Hennig et al., 2003;
Köhler et al., 2003)
(data not shown). Further studies will reveal which of these partners
participate(s) in CAF-1 complex-independent functions of MSI1 during plant
development.
CAF-1 is needed for normal cell cycle progression during endoreduplication
The DNA content of CAF-1 mutant cells was increased in light- and
dark-grown seedlings. In both cases, DNA endoreduplication occurs during
normal development, but many mutant cells continued DNA endoreduplication for
on average one extra round. This observation was surprising considering that
RNAi knock-down of human p150 causes an accumulation of cells in S-phase
(Hoek and Stillman, 2003).
Because Arabidopsis CAF-1 mutants are viable, plant cells appear to
be less dependent on CAF-1 function, thus revealing an additional role of
CAF-1, i.e. to prevent excessive rounds of DNA endoreduplication. In yeast and
humans, CAF-1 appears to be required for kinetochore formation
(Sharp et al., 2002;
Sharp et al., 2003).
Interfering with kinetochore formation could potentially bypass mitosis and
cause an increased DNA content. Alternatively, failure to exit the G2 phase of
the cell cycle could cause an accumulation of cells with increased DNA
content. Because the effects on DNA content were similar in the strong
fas1-1 and fas2-4 alleles and in the weak fas1-4
and msi1-as alleles, even a partial loss of CAF-1 activity can
considerably affect cellular DNA content. However, CAF-1 is not absolutely
essential for normal cell cycle progression in Arabidopsis, as the
fas2-1 allele in Ler had a largely unchanged DNA content.
This suggests that the Ler accession contains genetic determinants
that make CAF-1 largely dispensable for normal cell cycle progression. Because
the developmental phenotypes of the two fas2 null alleles
fas2-1 and fas2-4 were similar in Ler and Col, only
the cell cycle function but not the developmental function of CAF-1 can be
compensated in Ler. In addition, this suggests that the developmental
defects of CAF-1 mutants are not a direct consequence of defective cell cycle
progression.
A role of CAF-1 in duplication of epigenetic information
CAF-1 has nucleosome assembly activity and functions by depositing histone
H3-H4 dimers onto newly synthesized DNA, as it is recruited to places of DNA
synthesis by PCNA (for a review see,
Loyola and Almouzni, 2004). As
CAF-1 facilitates replication-coupled chromatin assembly, it may be needed to
faithfully duplicate epigenetic information in chromatin during mitosis
(Enomoto and Berman, 1998;
Ridgway and Almouzni, 2000;
van Nocker, 2003;
Henikoff et al., 2004).
Indeed, CAF-1 is needed for the maintenance of gene silencing in yeast,
mammals and plants (Enomoto and Berman,
1998; Tchenio et al.,
2001; Takeda et al.,
2004; Ono et al.,
2006; Schönrock et al.,
2006b). In Arabidopsis, CAF-1 contributes to the
compaction of heterochromatin but is not essential for the silencing of most
heterochromatic genes (Schönrock et
al., 2006b). Nevertheless, several silent heterochromatic loci
become stochastically activated in some cells in a fraction of fas2
plants (Ono et al., 2006). In
addition, the euchromatic GLABRA2 gene has a cell-type-specific
chromatin environment, which is lost in fas2 mutants
(Costa and Shaw, 2006).
Interestingly, plants containing greatly reduced MSI1 levels suffer from a
progressive loss of floral morphology, whereas none of the analyzed
fas1 or fas2 mutant alleles showed a similar increase in
phenotype severity (Hennig et al.,
2003). In contrast to the morphological phenotype, the molecular
phenotype of fas2 mutants increased with plant age as the expression
of SCR::GFP was disturbed more in older than in younger roots
(Kaya et al., 2001), and the
percentage of plants with de-repressed CACTA transposons increased
from 20% to 65% (Ono et al.,
2006). Some of the pleiotropic phenotypes of CAF-1 mutants occur
stochastically (Leyser and Furner,
1992; Kaya et al.,
2001) and are most likely caused by stochastic defects in the
expression of developmental regulators. Thus, CAF-1 might be needed for normal
development because it facilitates the faithful duplication of epigenetic
information in chromatin during mitosis.
CAF-1 is required for the control of multiple developmental processes in plants
In yeast, double mutants defective in CAF-1 and HIR or ASF1 cause defects
in cell proliferation (Tyler et al.,
1999; Sharp et al.,
2001; Sharp et al.,
2002), and CAF-1 mutants in higher eukaryotes, including
Arabidopsis, most likely suffer from delayed cell division as well
(Hoek and Stillman, 2003;
Schönrock et al., 2006b).
Several of the less stochastic phenotypes of Arabidopsis CAF-1
mutants, including changes in embryonic and post-embryonic organ size (e.g.
hypocotyl, rosette), and reduced growth rates, could be caused by defects in
cell proliferation. Because CAF-1 is needed for both efficient S-phase
progression (Hoek and Stillman,
2003) and mitosis (Sharp et
al., 2002; Sharp et al.,
2003), absence of CAF-1 activity can interfere with steps in
development that are based on a sensitive balance between cell cycle
progression, cell division and differentiation. In meristems, this balance is
particularly important, and mutant phenotypes arising from imbalances in
meristems are therefore very obvious. Our results demonstrate, however, that
the function of CAF-1 is not restricted to meristems, but is also required
during many other differentiation and developmental processes. Most
strikingly, even the differentiation of single-celled trichomes, which is
independent of cell division, depends on CAF-1 activity.
Supplementary material
Supplementary material for this article is available at
http://dev.biologists.org/cgi/content/full/133/21/4163/DC1
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ACKNOWLEDGMENTS |
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REFERENCES |
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