Department of Botany, Division of Life Sciences, University of Toronto, 1265 Military Trail, West Hill, Ontario M1C 1A4, Canada
* Author for correspondence (e-mail: riggs{at}utsc.utoronto.ca)
Accepted 10 April 2003
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SUMMARY |
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Key words: Condensation, Meristem, Meiosis, Fasciation, Arabidopsis thaliana, SMC2, Gametogenesis
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INTRODUCTION |
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The SMC proteins are evolutionarily conserved across phylogenetic kingdoms
with representatives in both prokaryotes and eukaryotes. The eukaryotic SMC
proteins can be subdivided into six distinct subfamilies (SMC1-6)
(Hirano, 2002;
Jessberger, 2002
), and three
functional categories can be defined based on the formation of their
heterodimers. The SMC2/SMC4 heterodimers assemble with three other non-SMC
proteins to form the `condensin' complex, which is involved in mitotic
chromosome condensation and dosage compensation
(Hirano and Mitchison, 1994
;
Lieb et al., 1998
;
Sutani et al., 1999
;
Kimura et al., 2001
). Members
of the SMC1 and SMC3 families form heterodimers to give rise to the `cohesin'
complex, which is involved in sister chromatid cohesion
(Michaelis et al., 1997
;
Losada et al., 1998
). Lastly,
SMC5 and SMC6 members form heterodimers that are involved in DNA
recombination/repair (Mengiste et al.,
1999
; Fousteri and Lehmann,
2000
). All SMC proteins consist of five distinct domains:
nucleotide-binding domains located at the N and C termini lie adjacent to two
coiled-coil regions, which are separated by a hinge region
(Hirano, 2002
;
Jessberger, 2002
). The
catalytic function of the SMC subunits is regulated by non-SMC members of
these multisubunit complexes. Mutations in genes encoding components of SMC
complexes result in a variety of defects, ranging from lethality to
developmental abnormalities (Saka et al.,
1994
; Strunnikov et al.,
1995
; Bhat et al.,
1996
; Lieb et al.,
1998
; Freeman et al.,
2000
; Steffensen et al.,
2001
; Bhalla et al.,
2002
; Hagstrom et al.,
2002
).
In both plants and animals, alterations in chromatin structure and dynamics
are implicated as factors controlling both cell cycle progression and aspects
of development (reviewed by Muller and
Leutz, 2001; Reyes et al.,
2002
; Berger and Gaudin,
2003
). Whereas in many metazoans mutations in these genes are
lethal, orthologous mutants in plants are often tolerated, resulting in
developmental and/or physiological defects
(Wagner, 2003
). For example,
plants harboring an antisense construct of histone deacetylase 1
(AtHD1) exhibit pleiotropic defects in growth and development, and
some of these could be correlated with ectopic expression of the
SUPERMAN gene (Tian and Chen,
2001
). Similarly, disruption of the topoisomerase I gene leads to
phyllotaxy defects and bifurcation of lateral shoots
(Takahashi et al., 2002
).
Arabidopsis plants deficient in telomerase function also exhibit
developmental abnormalities with grossly enlarged meristems, altered leaf
morphology and low viable seed set (Riha
et al., 2001
). Lastly, mutations in chromatin assembly factor
(CAF1) subunits, which assemble nucleosomes onto nascent DNA, underpin the
fasciata phenotype (Kaya et al.,
2001
).
Despite significant progress in our understanding of molecular and
biochemical aspects of chromatin remodeling and chromosome condensation in
yeast and in animal systems, very little information exists in plants.
Arabidopsis affords an excellent system to explore these basic
cellular processes as the genome has been sequenced and annotated, genetic
manipulations are facile, and there exists a large collection of insertion
mutant lines for reverse genetics studies. We therefore sought to identify
mutations in Arabidopsis condensin genes. Here we report the
characterization of two Arabidopsis SMC2 genes, AtCAP-E1 and
AtCAP-E2 (Arabidopsis thaliana Chromosome Associated Protein
subunit E, a nomenclature that conforms to the designation given to SMC2
orthologs in higher eukaryotes). Unlike other characterized diploid organisms,
Arabidopsis contains two members of the SMC2 family. Recently, a
T-DNA insertion into AtCAP-E1 was reported to be responsible for the
titan3 mutant, which exhibits enlarged endosperm nuclei and aberrant
mitotic figures but otherwise develops normally and is fecund
(Liu et al., 2002). Given the
essential role for SMC2 proteins in chromosome condensation in other
organisms, it could be predicted that disruption of SMC2 function would have
profound effects on cell division and plant development. We identified a
mutant of AtCAP-E2, which exhibits no obvious developmental defects,
but the AtCAP-E1-/-, AtCAP-E2-/-
double mutant results in embryo lethality. Transgenic plants expressing an
antisense construct exhibited reduced levels of both SMC2 transcripts, and
pleiotropic phenotypes including slow growth, disorganized meristems, and
fasciation. Our data indicate that proper developmental patterning requires a
threshold level of SMC2 proteins to support condensin complex function.
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MATERIALS AND METHODS |
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Yeast complementation
The AtCAP-E1 cDNA was digested with NotI and
SpeI and ligated into the NotI and XbaI sites in
pYES2. Transformation of smc2-6 cells was performed
according to the method of Gietz and Woods
(Gietz and Woods, 1998
).
Transformants were selected at the restrictive temperature (37°C) on DOB
dropout base plus CSM lacking uracil containing 2% D-galactose. For
4',6-diamidino-2-phenylindole (DAPI) staining, liquid cultures [wild
type (YPH499), smc2-
6, and transformed cells] were
grown overnight at 30°C and then shifted to 37°C for 2 hours. An
aliquot of cells was harvested, fixed in 2.5% glutaraldehyde, rinsed, and
stained with 2 µg/ml DAPI solution. DAPI staining was monitored using a
Zeiss axiophot microscope.
RNA isolation and RT-PCR analysis
Total RNA was prepared from Arabidopsis roots, stems, mature
leaves, floral buds and mature green siliques using the TRIZOL (Gibco)
reagent. RT-PCR was performed as follows. Forward
(5'-GATACATGCAAAGATGAAGGAATG-3') and reverse
(5'-TATCATTCTTCCTATGTTCTGTGTGTG-3') primers were used to amplify a
762 bp fragment from both AtCAP-E1 and AtCAP-E2.
First-strand cDNA synthesis was performed with 1 µg of total RNA and the
backward primer in a 25 µl reaction according to the 5'RACE protocol
(Gibco). PCR was performed by making tenfold serial dilutions (10-1
and 10-2) starting from the same amount of first-strand cDNA. The
volume of each tenfold RT-dilution step used as a template was 6% of the final
PCR volume. For CAPS analysis (Konieczny
and Ausubel, 1993), PCR products were digested with SspI
or XbaI and DNA gel blotting was performed with the 762 bp cDNA
fragment as the probe.
Construction of AtCAP-E1 promoter::ß-glucuronidase gene
and plant transformation
A 2.7 kb region upstream of the AtCAP-E1 start codon was amplified
by PCR with the primers: 5'-CGGGATCCAGCAATAGCTTTAGCTTTCGCC-3' and
5'-TCCCCCGGGCTTCCTTCTTCTTCTTCCCC-3'. The amplified fragment was
ligated into pBI101.2 (Jefferson et al.,
1987) upstream of the ß-glucuronidase gene. The construct was
mobilized into Agrobacterium (GV3101) and used to transform
(Landsberg erecta ecotype) plants by the floral dip method
(Clough and Bent, 1998
).
Transformants were selected on Murashige and Skoog (MS) plates (1x MS
salts pH 5.7, 3% sucrose, 0.5 g/l MES, 0.8% phytagar) containing 50 µg/ml
kanamycin. The histochemical assay for GUS was performed as described
previously (Jefferson et al.,
1987
).
In situ hybridization
In situ hybridization was performed essentially as described by Jackson
(Jackson, 1991). A 196 bp
region of the Arabidopsis histone H4 gene was labeled with
digoxigenin. In vitro transcription was performed following the manufacturer's
(Roche) protocol. Wild-type and antisense seedlings were fixed in FAA (3.7%
formaldehyde, 5% acetic acid, 50% ethanol) for 2 hours, dehydrated in an
ethanol/Histoclear series, embedded in TissuePrep and sectioned at 10 µm.
After the color development, sections were mounted in VectaShield mounting
medium (Vector Laboratories, USA) and photographed using a Zeiss axiophot
microscope and digital imaging system.
Isolation of an AtCAP-E2-/- mutant
A BLAST search in the SAIL (Syngenta Arabidopsis Insertion Library)
database with the AtCAP-E2 genomic sequence (AGI identifier
At3g47460) revealed a line (36-C09) with a T-DNA insertion near the 3'
end of the gene. Seeds of this line were germinated on MS plates containing
glufosinate ammonium (BASTA) to select for plants having a T-DNA insertion.
The integration site of the T-DNA was determined by PCR amplification and
sequencing of a 1061 bp fragment using a gene-specific primer (5'
GATACATGCAAAGATGAGGAATG-3') and the T-DNA left border primer
(5'-TAGCATCTGAATTTCATAACCAATCTCGATACAC-3'). The genotype of the
individual plants grown on BASTA was determined by two sets of PCR reactions.
The first used gene-specific primers flanking the T-DNA
(5'-GATACATGCAAAGATGAAGGAATG-3';
5'-TATCATTCTTCCTATGTTCTGTGTGTG-3') to identify the presence of a
wild-type allele. Plants heterozygous for the T-DNA insertion gave rise to a
1491 bp AtCAP-E2 gene fragment with PCR primers flanking the T-DNA
insertion site, while no band corresponding to the AtCAP-E2 locus was
amplified from the homozygous plants. The second PCR employed a gene-specific
primer and the T-DNA left border primer to confirm the presence of a mutant
allele.
Generation of double mutants and analysis of F2
progeny
Double mutant plants were generated by crossing homozygous
AtCAP-E1-/- (ttn3 mutant; Wassilewskija ecotype;
WS) with AtCAP-E2-/- (Columbia ecotype; Col) plants.
F1 seeds were germinated on medium containing both BASTA and
kanamycin and 100% resistance to both antibiotics was observed (The T-DNA
insertion at the AtCAP-E1 locus confers kanamycin resistance while
the T-DNA insertion at the AtCAP-E2 locus confers BASTA resistance).
F2 seedlings were selected on MS plates containing both
antibiotics, and 180 F2 seedlings were genotyped by employing three
sets of primers. Based on the site of insertion of the T-DNAs, the first
primer pair (5'-GATACATGCAAAGATGAAGGAATG-3';
5'-TATCATTCTTCCTATGTTCTGTGTGTG-3') was chosen to flank the
insertion sites and amplification of 1.65 kb (amplified from the
AtCAP-E1 locus) and 1.49 kb (amplified from the AtCAP-E2
locus) fragments are indicative of a wild-type allele. Amplification of a
single band (either 1.65 or 1.49) represented heterozygosity at the given
locus and homozygosity for a T-DNA insertion in the other gene. The other sets
of primer pairs employed an allele-specific primer together with a T-DNA
primer to test for the presence of a T-DNA insertion as described in the
previous section.
Because the two single mutants are in different genetic backgrounds (WS and Col) we crossed these wild-type plants to determine if any inter-ecotype incompatibilities exist that would compromise development and/or fecundity. We observed no evidence of ovule or embryo abortion, which validates the phenotypic defects observed in the F2 progeny of a cross between the single mutants. Genetic evidence supporting this contention is twofold. In siliques of E1+/-, E2+/- plants, the number of aborted ovules and aborted embryos conformed to the ratios expected based on the parental genotype (data not shown) such as they did in E1+/-, E2-/-. Secondly, the anaphase bridges observed in AtCAP-E1+/-, AtCAP-E2-/- pollen mother cells were not observed in AtCAP-E1+/-, AtCAP-E2+/- pollen mother cells.
Generation of antisense transgenic plants
Arabidopsis plants (Landsberg erecta ecotype) carrying
the AtCAP-E1 cDNA in an antisense orientation were generated by
inserting a 2.3 kb fragment of the AtCAP-E1 cDNA in the reverse
orientation in pBI121, such that transcription of the antisense strand was
driven by the CaMV-35S constitutive promoter. Transgenic plants were generated
as described above. All plants were grown at 25°C under a 16-hour
light/8-hour dark regime.
Histological analysis
Siliques were dissected after fixation in an ethanol:acetic acid (3:1)
solution, cleared overnight in chloral hydrate:glycerol:water (8:2:1) and
imaged by Nomarski optics.
For DAPI staining of pollen mother cells, fresh anthers were dissected on a slide in 40 µl of 10 mM sodium citrate buffer pH 4.0 using a needle under a dissecting misroscope. After releasing the contents of the anthers the sample was mounted in 15 µl of Vectashield containing DAPI. After the addition of a coverslip, the slides were viewed by fluorescence using a Zeiss Axiophot microscope.
For Toluidine Blue and DAPI staining of SAMs, 8-day-old wild-type and 18-day-old antisense seedlings were fixed in FAA, embedded in wax, and 8 µm sections were cut. Images were captured with a Zeiss axiophot microscope equipped with a digital image capture system.
For scanning electron microscopy (SEM), seedlings were fixed in FAA, dehydrated, critical point dried, and coated with gold particles. Specimens were viewed using a Hitachi S-2500 microscope.
Wild-type and antisense seedlings were grown vertically on agar plates for 10 days for confocal laser scanning microscopy (CLSM). For Aniline Blue staining, roots were fixed in a 2.5% NaOH/0.1% sodium dodecyl sulfate solution for 18 hours at room temperature, rinsed in water, and dehydrated by passing through an ethanol series. The tissue was stained for 4 hours with 0.4% Aniline Blue. After staining, tissue was dehydrated to 100% ethanol and cleared by passing through a 3:1, 1:1 and 1:3 series of ethanol:methyl salicylate solution followed by two washes in 100% methyl salicylate. Median longitudinal optical sections of the root tip were obtained using an Olympus Fluoview (FV5-PSU) CLSM.
Immunoblotting
A 1302 bp DraI to EcoRI fragment of the AtCAP-E1
cDNA was cloned between SmaI and EcoRI sites of pGEX2TK
(Invitrogen) vector downstream of the glutathione S-transferase (GST) fragment
to produce a GST-tagged recombinant protein in bacteria. The recombinant
protein was purified and used to immunize rabbits. The affinity-purified
polyclonal antibodies were diluted 1:2500 for immunoblotting with total
protein extracts prepared from 7-day-old seedlings.
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RESULTS |
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AtCAP-E1 complements the yeast
smc2-6 mutant
To confirm that the AtCAP-E1 gene encodes a functional SMC2
protein, we used it to transform the yeast SMC2 mutant
smc2-6, which shows a temperature-sensitive defect in
chromosome condensation and segregation
(Strunnikov et al., 1995
).
Mutant cells undergo cell-cycle arrest at the non-permissive temperature
(37°C). Some of these cells exhibit the classical `cut' phenotype
wherein the cells have a fully elongated mitotic spindle but their chromosomes
fail to separate and appear as a stretched mass of DNA between the two
daughter cells. This nuclear morphology is indicative of a defect in
chromosome condensation.
We transformed smc2-6 cells with the
AtCAP-E1 cDNA under the control of the galactose-inducible GAL1
promoter and selected for transformants in the presence of galactose at
37°C. Transformed cells were streaked on plates along with the wild-type
and smc2-
6 mutant cells in the presence and absence
of galactose and incubated at 37°C. Transformed
smc2-
6 cells grew to nearly wild-type levels at
37°C in the presence of galactose (Fig.
1B), but failed to grow in the absence of galactose
(Fig. 1C). Fig. 1D-G shows the morphology
of the DAPI-stained chromatin by fluorescence microscopy. The stretched
nuclear DNA in the smc2-
6 mutant cells and
smc2-
6 cells transformed with AtCAP-E1 cDNA,
in the absence of galactose, can be seen in
Fig. 1D and E, respectively.
Normal chromosome segregation was observed in wild-type cells
(Fig. 1F), and transformed
cells (Fig. 1G) when grown in
the presence of galactose.
|
Differential expression of AtCAP-E1 and
AtCAP-E2
The Arabidopsis genome contains two SMC2 family members, encoding
AtCAP-E1 and AtCAP-E2, which are 90% similar at the protein
level. In order to examine the expression profile of both genes, we conducted
RT-PCR followed by CAPS analysis (see Materials and Methods). Primers that
exactly matched two regions in both genes were used in RT-PCR reactions, and
thus the PCR product pool represented transcripts from both genes. The
AtCAP-E1 and AtCAP-E2 mRNAs were most abundant in roots and
young floral buds. The expression was intermediate in stems and low in fully
developed leaves and mature siliques (Fig.
2A).
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To study the spatial and temporal expression of the AtCAP-E1 gene, an AtCAP-E1 promoter::ß-glucuronidase (GUS) reporter chimeric gene was constructed and used to generate transgenic plants. Several independent transformants were assayed histochemically for GUS activity, and all primary transformants exhibited the same qualitative expression pattern.
In plants, apical meristems and lateral organ primordia are sites of active
cell division. Fig. 2E shows
GUS staining of the apex of a young seedling, wherein the entire apical dome
and the emerging true leaves are stained. The root apical meristem (RAM) and
emerging lateral root primordia also stain intensely
(Fig. 2F,G). No activity was
observed in differentiated root tissue. In mature plants, strong GUS
expression was observed in newly formed organs. Uniform GUS expression was
detected during early leaf development, which declined in a basipetal gradient
starting from the distal region of immature leaves first
(Fig. 2H). The onset of
mesophyll cell expansion starts at the leaf tip and progresses towards the
leaf base forming a gradient of cells at different developmental stages
(Pyke et al., 1991). Thus, a
decline in GUS activity as development proceeds can be correlated with the
decline in mitotic activity. Fig.
2I shows that AtCAP-E1 is strongly expressed in
developing floral buds, which are undergoing active cell division. After organ
formation, strong GUS activity was observed in all four floral whorls and
staining in young anthers and the gynoecium suggested that the SMC2 gene(s)
might be active during meiosis (data not shown). Owing to the long half-life
of GUS and the brief duration of meiosis in Arabidopsis, it was not
possible to be certain if the GUS staining observed in the pollen mother cells
(PMCs) was generated during or just prior to meiosis. Therefore, we elected to
do RNA in situ hybridization using cross sections of whole inflorescences to
precisely define the temporal expression pattern of AtCAP-E1.
Expression in stamens was observed in the PMCs and in the surrounding tapetal
cells during premeiotic S phase, prophase I, and anaphase I stages of meiosis
(data not shown, but see below).
Identification and characterization of SMC2 insertion
mutants
We undertook a reverse genetics approach to find mutants in the two
Arabidopsis SMC2 genes. The Arabidopsis Biological Resource Center
(ABRC) T-DNA lines were screened to identify insertions into AtCAP-E1
and we found one pool containing such an insertion. During the course of this
work, Liu et al. (Liu et al.,
2002) reported that the titan3 mutant was due to an
insertion into this gene, and hence we discontinued characterization of the
ABRC line, using the available ttn3 mutant for additional studies. A
T-DNA insertion mutant in the AtCAP-E2 gene was identified in the
Syngenta T-DNA mutant collection (McElver
et al., 2001
). Sequencing revealed that the insertion occurred at
the beginning of the 17th exon. Semi-quantitative RT-PCR revealed no
AtCAP-E2 mRNA in 7-day-old AtCAP-E2-/- seedlings
(data not shown). To determine the copy number of T-DNA insertions, seeds from
the segregating population were grown on BASTA and resistant seedlings were
counted (1399 resistant versus 498 sensitive). This ratio closely matched with
the expected 3:1 ratio suggesting a single T-DNA insertion. Southern blotting
also confirmed that AtCAP-E2-/- plants contained a single
T-DNA insertion (data not shown).
We could not identify any obvious phenotype associated with the AtCAP-E2 mutation when the plants were grown under standard conditions.
Double mutants of AtCAP-E1 and AtCAP-E2 result in
embryo lethality
Since ttn3 (AtCAP-E1-/-) plants exhibited a
subtle endosperm phenotype with viable embryos and no post-embryonic
phenotype, and the AtCAP-E2-/- plants demonstrated no
obvious defects, we performed genetic crosses between the two homozygous
single mutant plants to generate double mutants. The expected relative
abundance of each F2 genotype is given in
Table 1. Both mutants are the
result of T-DNA insertions: E1 (referring to insertion in AtCAP-E1)
is marked by an insertion of a kanamycin resistance gene while E2 (referring
to insertion in AtCAP-E2) contains a BASTA resistance gene. Thus,
E1- alleles and E2- alleles would confer resistance to
kanamycin and BASTA, respectively (Table
1, column 2). To enrich for double mutants, F2 seeds
were germinated on media containing both antibiotics.
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The E1-/-E2-/-, E1-/-E2+/-, and E1+/-E2-/- diploid genotypes all require that at least one of the gametes has the haploid genome E1-E2-. Since the three aforementioned genotypes were either missing or under-represented in the F2 generation, it raised the possibility that the haploid spores with the E1-E2- genotype could not successfully complete gametogenesis. To test this hypothesis we performed reciprocal crosses of wild type with double heterozygotes, E1+/-E2+/-, and scored the F1 seeds for their ability to grow on the kanamycin/BASTA selection media. The types of haploid genotypes that can be produced by the double heterozygote are E1+E2+, E1+E2-, E1-E2+, and E1-E2-. Since the wild-type parent only produces the E1+E2+ (which is sensitive to both kanamycin and BASTA), only the F1 offspring that receive the E1-E2- genotype from the double heterozygous parent would be able to grow on the selection medium. Thus only 25% of the F1 plants would be expected to be resistant to both kanamycin and BASTA. When the E1+/-E2+/- plants were used as the male parent only 3.2% of the F1 seedlings (n=248) were resistant to both antibiotics. Thus, the greatly reduced number of E1+/-E2+/- individuals indicates that only 13% (3.2% observed versus 25% expected) of the expected number of E1-E2- pollen grains are able to successfully complete male gametogenesis. Similarly, in the reciprocal cross in which the E1+/-E2+/- plants were used as the female parent, only 12.5% of the F1 seedlings (n=202) were resistant to both antibiotics. This indicates that there is a 50% reduction in the ability of the megaspore to complete female gametogenesis. Thus, failure during gametogenesis in E1-E2- gametes is a major, but not exclusive source of lethality.
To further investigate the issue of viability we examined the contents of the siliques. An indicator of early lethality in Arabidopsis is a paucity of normal seeds in developing siliques. Wild-type and the single mutant plants yielded normal siliques filled with full, green seeds (Fig. 3A-C). However, severe seed defects were observed in the mature siliques of the self-crossed E1+/-E2-/- individuals (Fig. 3D). Among the 26 green siliques that we examined, we observed 43.43% normal green seeds, 30.86% discolored and mis-shapened seeds, and 25.7% unfertilized ovules, which appeared as empty spaces (aborted ovule development presumably associated with failure to complete female gametogenesis) in the silique. We used differential interference contrast microscopy (DIC) to determine the developmental stage at which the embryos in these abnormal seeds were arrested. The normal-appearing green seeds contained normal embryos at the globular to torpedo stages (Fig. 3E). Embryo development clearly differed in the abnormal sibling seeds with embryos arrested at the globular stage of embryogenesis (Fig. 3F). The seeds harboring the mutant embryos were usually smaller. Occasionally, we observed mutant embryos at the late globular stage with distinct defects in cell patterning of the suspensor as well as the embryo proper. The suspensors of these embryos consisted of randomly arranged multi-cell layers with both transverse and longitudinal cell division planes, whereas the suspensors of wild-type embryos consist of single files of six to 11 cells resulting from a series of transverse divisions. Similarly, the abnormal embryos exhibited an altered morphology probably due to atypical cell division planes, resulting in an arrested globular-like embryo (Fig. 3G,H). Thus, our silique analyses show that self-crosses of E1+/-E2-/- individuals often produce many abnormal seeds containing aborted embryos and some seeds fail to form at all.
|
Meiotic segregation defects in AtCAP-E1+/-,
AtCAP-E2-/- plants
The E1+/-E2-/- genotype was the only genotype we
recovered that carries only one functional SMC2 allele. Plants of this
genotype often exhibit vegetative abnormalities (see the next section), and we
examined meiosis in these plants to assess chromosome condensation/segregation
defects. In plants, meiosis punctuates the sporophytic and gametophytic phases
of the life cycle. Male meiosis (microsporogenesis) occurs in the pollen
mother cells (PMCs) that give rise to four haploid microspores, each of which
undergoes mitosis and differentiates into a mature pollen grain. Our GUS
reporter gene studies as well as the in situ hybridization experiments
indicated that the SMC2 genes are expressed in PMCs, and this fact prompted us
to ascertain whether there exists an aberrant chromatin phenotype during
meiosis in E1+/-E2+/- and
E1+/-E2-/- plants. During meiosis I, chromatin
condensation reaches a maximal level at metaphase I/anaphase I and hence we
examined PMCs undergoing these two stages during meiosis I. In wild-type PMCs,
congression of discrete bivalents was observed, and later, at the onset of
anaphase, the chromosomes segregated properly
(Fig. 4A,D). Cytological
analysis of DAPI-stained PMCs of E1+/-E2+/- plants
showed no aberrant chromatin behavior during meiosis I, and normal homologous
chromosome segregation was observed (data not shown). In contrast, PMCs of
E1+/-E2-/- plants often contained diffuse and
non-discrete bivalents at metaphase I (Fig.
4C), and exhibited regions of stretched chromatin bridges during
anaphase I with less condensed chromosomes
(Fig. 4F). Nonetheless, these
chromosomes appeared more condensed than the interphase chromatin as observed
by DAPI staining (data not shown). Thus, this meiosis I defect mimics those
observed in other condensin mutants during mitosis in yeast
(Saka et al., 1994;
Strunnikov et al., 1995
;
Bhalla et al., 2002
) and
Drosophila (Bhat et al.,
1996
; Steffensen et al.,
2001
) and during mitosis and meiosis II in C. elegans
(Hagstrom et al., 2002
).
|
Although the antisense seedlings germinated with no obvious embryonic
defects, pleiotropic phenotypes affecting various stages of post-embryonic
development were observed. The transcript levels of both AtCAP-E1 and
AtCAP-E2 in these seedlings were dramatically reduced
(Fig. 5A-C), confirming a
strong antisense suppression of the in vivo AtCAP-E1 and
AtCAP-E2 transcript pool. In the most severe cases, the shoot apical
meristem (SAM) developed into an enlarged dysfunctional mass
(Fig. 5E,F) and asymmetric
leaf-like structures often formed from the primary SAM
(Fig. 5G). However, these
plants usually produced axillary buds, which ultimately produced flowering
shoots that were developmentally delayed
(Fig. 6A,B). Typically, the
antisense plants produced shoots with varying degrees of stem and
inflorescence fasciation (Fig.
5H/I). Fasciation represents a disruption in the pattern of
organogenesis and is associated with breakdown or disruption in SAM structure
(Leyser and Furner, 1992),
resulting in altered leaf and floral phyllotaxy, and broadening, flattening,
and in extreme cases, bifurcation of the stem. Interestingly, stem and floral
fasciation was also observed in some E1+/-E2-/- plants
(Fig. 5J,K).
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Dosage effect of SMC2 proteins
We used immunoblotting to assess the SMC2 protein levels in wild type and
in different mutant backgrounds, using a polyclonal antiserum raised against a
fragment of AtCAP-E1. The antiserum detects a single protein of approximately
130 KDa in a total protein extract from 7-day old wild-type seedlings
(Fig. 9, lane1). Based on
conceptualized translation, this corresponds well with the size expected for
AtCAP-E1 and AtCAP-E2. In antisense seedlings, the signal is reduced
dramatically compared to wild type (lanes 2-4). In
E1-/-E2+/+ plants (titan 3), no band was
detected confirming that these plants did not contain detectable levels of
AtCAP-E1 protein (lane 5). Although AtCAP-E2 should be present in this
extract, its levels are too low to be detected by immunoblotting. A faint band
was detected from these extracts when six- to eightfold more protein extract
was used for immunoblotting (data not shown), suggesting that the sensitivity
of immunoblotting is most likely the limiting factor in detecting AtCAP-E2.
The protein band detected in the E1+/+E2-/- extract is
similar in strength to the wild type (lane 6). Extracts of
E1+/-E2-/- plants gave rise to a signal, which was
reduced relative to wild type, but significantly more than that of antisense
plants (lane 7), pointing to a significant knock-down of both
AtCAP-E1 and AtCAP-E2 gene products in the antisense plants.
Taken together, our data suggest that Arabidopsis plants can use both
proteins redundantly, that a reduction in SMC2 levels gives rise to fasciated
plants, and that a threshold level of SMC2 molecules is required for normal
embryogenesis and development.
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DISCUSSION |
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It is also possible that protein activity is controlled by
post-translational modifications. In S. pombe, the condensin complex
is translocated to the nucleus after the phosphorylation of the SMC4 ortholog
by the cdc2 mitotic kinase
(Sutani et al., 1999). In
Xenopus and humans, phosphorylation of a non-SMC component of the
complex by cdc2 has been implicated in supercoiling and condensation
activities (Kimura et al.,
1998
; Kimura et al.,
2001
), and in C. elegans, AIR-2/AuroraB kinase-dependent
phosphorylation is required for the cell cycle-dependent localization of the
condensin complex (Hagstrom et al.,
2002
). We are currently employing immunocytochemistry to address
questions of cell cycle-dependent expression and localization of SMC2 proteins
using synchronized suspension cultured cells.
How do reduced levels of SMC2 influence viability?
In several systems, in vitro experiments have demonstrated that the
condensin complex possesses a DNA-dependent ATPase activity that induces
positive supercoiling, which probably results in a reorganization of the
topology of DNA loop domains (Kimura et
al., 1999; Kimura et al.,
2001
). In Drosophila, mitotic defects in embryos and
formation of chromatin bridges are caused by mutations in the BARREN
gene, which encode a non-SMC condensin subunit
(Bhat et al., 1996
). Similarly,
a complete or partial depletion of SMC2/SMC4 transcripts by RNAi treatment in
C. elegans, results either in embryo lethality or in mature adults
with a variety of developmental defects, respectively
(Hagstrom et al., 2002
).
Analysis of the mutant progeny revealed chromosome segregation defects in both
somatic and germline cells, and as we observed in Arabidopsis,
affected cells were abnormally shaped and fecundity was compromised. Thus, in
the absence of a functional condensin complex, chromatin may remain relatively
decondensed and tangles may remain unresolved, leading to chromosome breakage
and abnormal chromosome segregation.
We observed that the highest levels of Arabidopsis SMC2 transcripts occur in tissues with high mitotic indices. We were interested in determining if reduced levels of SMC2 protein also affected meiotic chromosome behavior. During meiosis, two distinct nuclear divisions occur to reduce the chromosome number; in the first, homologous chromosomes segregate, while at meiosis II, sister chromatids segregate.
Our GUS and in situ hybridization studies of wild-type plants demonstrated
that SMC2 genes are expressed in meiotic cells. But from these studies it was
not possible to determine if the SMC2 protein plays a role in the first or
second meiotic division, or both. We were not able to recover either
E1-/-E2-/- or E1-/-E2+/- mutants
due to lethality early in development. However, we did recover a limited
number of E1+/-E2-/- plants and examined their
chromosome behavior in the first division. These plants showed a reduction in
condensation at metaphase and anaphase I, and a mild cut-type
phenotype at anaphase I. In order for homologous chromosomes to segregate from
each other they must resolve the chiasmata, which hold them together.
Chiasmata resolution requires that sister chromatids decatenate from each
other along the chromosome length. Thus the cut-phenotype at meiosis
I is probably due to failure to completely decatenate sister chromatids. We
did not examine the second meiotic division, but given that the chromosomes
did not properly segregate at the first meiotic division, it would be
difficult to distinguish affects specific to the second division from
secondary effects due to failure at the first division. Our study is the first
report demonstrating a role for SMC2 proteins in the first meiotic division.
The only other report on the role of SMC2 proteins in meiosis focused on RNAi
depletion of the C. elegans SMC2 gene [Mix-1
(Hagstrom et al., 2002)].
These authors reported that depletion of Mix-1 in meiotic cells did
not affect first division segregation, but did affect meiosis II. Compared
with our observations, this contrasting result may be due to the fact that the
Mix-1/SMC4 condensin complex, unlike the condensin complexes
of other organisms, is not associated along the central axis of the
chromosome, but instead outlines condensing prophase chromosomes, then
localizes specifically to the centromere region.
How do reduced levels of SMC2 affect development?
Development of the aerial parts of plants relies on the activity of
meristems, which continuously initiate new organs. Functional meristems are
maintained by a coordinated balance between stem cell divisions at the summit
of the meristem and loss of cells by differentiation to form primordia at its
flanks. The meristem phenotypes we observed could be due to two separate, but
not exclusive, mechanisms. First, the morphogenetic consequences could be a
direct effect of an altered cell cycle. If chromatin remains relatively
decondensed and catenated, the propensity for DNA topological stress is
elevated in actively proliferating cells, and this may activate a checkpoint
control to result in cell cycle arrest. Indeed, very few cells of the enlarged
meristems in antisense plants contain histone H4 mRNA, indicating that most
cells have ceased cycling. Other studies involving the perturbation of cell
cycle regulatory factors have shown that these also can give rise to meristem
defects. Arabidopsis plants expressing a dominant-negative form of
cdc2a can exhibit defects in apical/basal patterning of the embryo,
and in some instances meristems elaborate deformed leaves in an incorrect
phyllotactic pattern (Hemerly et al.,
2000). Similarly, the overexpression of cyclin D3 results
in a variety of developmental defects, including meristem disorganization
(Riou-Khamlichi et al., 1999
).
In root initials, alterations in the activity of a CDK-activating kinase led
to differentiation of the initials, followed by cessation of cell division
(Umeda et al., 2000
).
The second potential mechanism involves epigenetic control of
morphogenesis. Evidence is accumulating that condensins and other factors
involved in DNA/chromatin dynamics epigenetically alter gene expression
programs. One of the regulatory proteins of the Drosophila condensin
subunit interacts with polycomb group proteins to maintain transcriptional
silencing of homeotic genes (Lupo et al.,
2001). This indicates that condensin might act in the epigenetic
control of gene expression. A similar mechanism has been suggested in the
fasciata CAF-1 mutant that ectopically expresses the WUSCHEL
and SCARECROW genes (Kaya et al.,
2001
). Molecular and cytological studies in Arabidopsis
have included analyses of centromeres and associated heterochromatin, and
surprisingly, these regions contain expressed genes, including some encoding
putative transcription factors and other signaling molecules
(Copenhaver et al., 1999
). If
the Arabidopsis condensin preferentially associates with
heterochromatin, it may dictate how and when genes located in heterochromatin
are expressed, and in antisense or mutant plants having reduced levels of
SMC2, this may lead to misregulation of gene expression. Whether the
developmental defects we observed are a direct consequence of chromosomal
damage or an indirect effect mediated by an epigenetic mechanism needs to be
addressed.
In antisense plants exhibiting severe phenotypes, the primary SAM enlarges
into a callus-like mass of tissue and aborts, but functional, albeit
fasciated, axillary meristems arise. The difference in sensitivity of the
primary and axillary meristems to impaired chromosome condensation in our
antisense plants could be due to low expression of the antisense gene from the
35S promoter in axillary meristems or a differential ability of the cells to
employ a checkpoint delay in response to defective chromosome segregation. It
is possible that the duration of the cell cycle is longer in axillary
meristems, which could allow more time for the cells to resolve the
chromosomes, allowing them to circumvent damage and proceed through an arrest
checkpoint. Although Laufs et al. (Laufs
et al., 1998) compared the mitotic indices and cell cycle dynamics
in inflorescence and floral meristems in Arabidopsis and found them
to be similar, to our knowledge no comparative information exists on cell
cycle parameters of primary versus axillary meristems.
Threshold levels of SMC2 are required for proper development
Amongst organisms in which SMC2 genes have been characterized,
Arabidopsis is unique in that it harbors two closely related genes
instead of one. Our experiments show that AtCAP-E1 and AtCAP-E2 can act
redundantly, as indicated by mild or no defects in the single mutants
(compared to the catastrophic consequences of the double null mutant), and the
similar expression patterns as gauged by RT-PCR. Although the qualitative
patterns of expression are similar, the two genes are differentially
expressed, with AtCAP-E1 transcripts accounting for most of the SMC2
transcript pool. Differential regulation of duplicated genes has been observed
in a number of species and probably drives functional divergence
(Pickett and Meeks-Wagner,
1995). Such differential regulation might ensure normal
development despite physiological or genetic perturbations. It is not known if
the duplication of SMC genes in Arabidopsis represents a unique
situation, or if plants in general have evolved this mechanism to avoid
problems associated with improper chromosome condensation.
Although both single mutants are relatively normal, heterozygosity for insertion into one gene in a null background for the second gene confers two distinct phenotypes: lethality during embryogenesis (E1-/-E2+/-) or fasciation of relatively normal plants (E1+/-E2-/-). This suggests that the effect of the SMC2 proteins is dose dependent. Reducing the AtCAP-E2 level to one half of its level in E1-/-E2+/+ plants, which develop normally, results in death during embryogenesis, and suggests that AtCAP-E2, despite its low levels in the cell, has a higher specificity for its target. In the antisense lines, where the expression of both genes is reduced, and in E1+/-E2-/- plants, fasciation occurs, indicating that a critical threshold level of SMC2 proteins are required to sponsor events of chromosome condensation and support normal morphogenesis. The use of SMC2 antibodies in chromatin immunoprecipitation assays may provide some clues on binding specificity and identify target sequences important for condensin complex function.
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ACKNOWLEDGMENTS |
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