The Plant Cell, Vol. 17, 2454–2472, September 2005, www.plantcell.org ª 2005 American Society of Plant Biologists
SWI3 Subunits of Putative SWI/SNF Chromatin-Remodeling
Complexes Play Distinct Roles during
Arabidopsis Development
W
wiez_ ewski,a Szymon Kaczanowski,a Yong Li,b
Tomasz J. Sarnowski,a,1 Gabino Rı́os,b,1,2 Jan Jásik,b,1 Szymon S
c
a
bia1,c Csaba Koncz,b
Aleksandra Kwiatkowska, Katarzyna Pawlikowska, Marta Kozbia1,c Piotr Koz
,c,3
a
and Andrzej Jerzmanowski
a Institute
of Biochemistry and Biophysics, Polish Academy of Sciences, 02-106 Warsaw, Poland
für Züchtungsforschung, D-50829 Köln, Germany
c Laboratory of Plant Molecular Biology, Warsaw University, 02-106 Warsaw, Poland
b Max-Planck-Institut
SWITCH/SUCROSE NONFERMENTING (SWI/SNF) chromatin-remodeling complexes mediate ATP-dependent alterations of
DNA–histone contacts. The minimal functional core of conserved SWI/SNF complexes consists of a SWI2/SNF2 ATPase,
SNF5, SWP73, and a pair of SWI3 subunits. Because of early duplication of the SWI3 gene family in plants, Arabidopsis
thaliana encodes four SWI3-like proteins that show remarkable functional diversification. Whereas ATSWI3A and ATSWI3B
form homodimers and heterodimers and interact with BSH/SNF5, ATSWI3C, and the flowering regulator FCA, ATSWI3D can
only bind ATSWI3B in yeast two-hybrid assays. Mutations of ATSWI3A and ATSWI3B arrest embryo development at the
globular stage. By a possible imprinting effect, the atswi3b mutations result in death for approximately half of both
macrospores and microspores. Mutations in ATSWI3C cause semidwarf stature, inhibition of root elongation, leaf curling,
aberrant stamen development, and reduced fertility. Plants carrying atswi3d mutations display severe dwarfism, alterations
in the number and development of flower organs, and complete male and female sterility. These data indicate that, by
possible contribution to the combinatorial assembly of different SWI/SNF complexes, the ATSWI3 proteins perform nonredundant
regulatory functions that affect embryogenesis and both the vegetative and reproductive phases of plant development.
INTRODUCTION
Chromatin-remodeling complexes (CRCs), which mediate ATPdependent alterations of DNA–histone contacts, provide essential links between signaling pathways and the chromatin-based
control of transcription, replication, repair, and recombination
(Klochendler-Yeivin et al., 2002; Martens and Winston, 2003;
Roberts and Orkin, 2004). Based on characteristics of their
SUCROSE NONFERMENTING2 (SNF2) family ATPase subunits,
the ATP-dependent CRCs are classified into SWITCH2 (SWI2)/
SNF2, IMITATION SWITCH (ISWI), Mi-2/ChromodomainHelicase-DNA binding protein (Mi-2/CHD), and INO80 subfamilies (Becker, 2002; Brzeski et al., 2003). SWI/SNF-like complexes
studied in yeast, Drosophila, and mammals constitute at least
nine subunits. The evolutionarily conserved core SWI/SNF
1 These
authors contributed equally to this work.
address: Departamento Bioquimica y Biologia Molecular,
Universidad de Valencia, Dr Moliner 50, 46100 Burjassot, Spain.
3 To whom correspondence should be addressed. E-mail andyj@ibb.
waw.pl; fax 4822-6584636.
The authors responsible for distribution of materials integral to the
findings presented in this article in accordance with the policy described
in the Instructions for Authors (www.plantcell.org) are: Tomasz
Sarnowski (tsarn@poczta.ibb.waw.pl) and Gabino Rı́os (gabino.rios@
uv.es).
W
Online version contains Web-only data.
Article, publication date, and citation information can be found at
www.plantcell.org/cgi/doi/10.1105/tpc.105.031203.
2 Current
subunits, corresponding to homologs of yeast Swi2p/Snf2p
ATPase, Snf5p, Swi3p, and Swp73p proteins, are essential
and sufficient to remodel chromatin in vitro (Phelan et al.,
1999; Sudarsanam and Winston, 2000).
In yeast, two ATP-dependent CRCs, SWI/SNF and Remodel
the Structure of Chromatin (RSC) (carrying Swi2p/Snf2p and
Sth1p ATPase, respectively), contribute to both transcriptional
activation and repression (Sudarsanam and Winston, 2000).
Mutations in genes encoding SWI/SNF subunits cause defects
in mating-type switch and sucrose fermentation and affect the
transcription of ;5% of yeast genes. The RSC complex influences many more genes and is essential for viability and progression
through mitosis (Cairns et al., 1996; Ng et al., 2002). In Drosophila,
a single ATPase (Brahma) is found in two different SWI/SNF- and
RSC-related complexes (BAP and PBAP) that show different
subunit composition and chromosomal distribution (Mohrmann
et al., 2004). As core subunits, these complexes carry the trithorax
group (trxG) Brahma (ATPase), Snr1 (Snf5p), and Moira (Swi3p)
proteins that are required for the maintenance of homeotic gene
expression patterns affecting oogenesis, embryogenesis, and
segmentation (Crosby et al., 1999). Human prototypes of mammalian SWI/SNF-like complexes, BAF (SWI/SNF-BAP type) and
PBAF (RSC/PBAP type), contain either the Brahma or BRG1
ATPase in complex with the INI1 (Snf5p), BAF170, and BAF155
(Swi3p type) core subunits. In various mammalian SWI/SNF complexes, these core subunits are combined with different regulatory
subunits, including histone deacetylase and retinoblastoma
Control of Development by SWI3 Proteins
tumor-suppressor Rb binding proteins (RbAP48). BRG1 and
human Brahma bind directly to Rb, regulating cell cycle progression. Mutations affecting core components of SWI/SNF
complexes lead to tumorigenesis in somatic tissues of mice and
humans, indicating their roles in tumor suppression (Roberts and
Orkin, 2004). Compared with late effects of Rb deficiency during
embryo development, mutations that inactivate BRG1 (but not
human Brahma), INI1 (Snf5p), and SRG3 (BAF155/SWI3) result in
early lethality during the blastocyte stage (Bultman et al., 2000;
Klochendler-Yeivin et al., 2000; Guidi et al., 2001; Kim et al., 2001).
Although no ATP-dependent CRCs have been characterized
to date in plants, comparative genome analyses indicate that
plants encode a remarkably high number of potential CRC
ATPase subunits (Reyes et al., 2002; Wagner, 2003). From 42
putative Arabidopsis thaliana SNF2-like ATPases (see the Plant
Chromatin Database at http://chromdb.org), 4 belong to the
canonical SWI2/SNF2 subfamily (Verbsky and Richards, 2001).
However, only one of these (Arabidopsis BRAHMA [At2g46020])
carries a C-terminal region resembling a bromodomain, a hallmark for binding acetylated Lys residues of histone tails (Hudson
et al., 2000; Brzeski et al., 2003). Genetic studies suggest both
positive and negative regulatory roles for members of the
Arabidopsis SNF2 gene family. Silencing of Arabidopsis
BRAHMA results in reduced fertility, curly leaves, homeotic
transformations during flower development, and photoperiodindependent early flowering by derepression of CONSTANS
(CO), FLOWERING LOCUS T (FT), and SUPPRESSION OF
OVEREXPRESSION OF CONSTANS1 (SOC1) (Farrona et al.,
2004). SPLAYED, which encodes a SNF2p/Sth1p-like ATPase
with an AT-hook motif (Wagner and Meyerowitz, 2002), is required together with LEAFY (LFY) and UNUSUAL FLORAL
ORGANS (UFO) for homeotic class B gene expression but also
acts as a repressor of floral transition under noninductive conditions. PHOTOPERIOD-INDEPENDENT EARLY FLOWERING1
(PIE1), encoding an ISWI-type ATPase (Noh and Amasino, 2003),
stimulates FRIGIDA-dependent expression of the floral suppressor FLOWERING LOCUS C (FLC). The pie1 mutation causes
early flowering under short days independent of FLC and acts as
a suppressor of petal defects caused by deficiency of the CURLY
LEAF Polycomb (PcG) factor, a repressor of AGAMOUS (Goodrich
et al., 1997). The CHD3-type ATPase PICKLE carries two copies of
a chromodomain for potential recognition of methyl-lysines in
histone tails and acts as a repressor of LEAFY COTYLEDON1,
a key activator of embryonic development (Ogas et al., 1999).
Mutations of INO80 affect the transcription of >100 genes and
reduce the frequency of homologous recombination (Fritsch et al.,
2004). A more distantly related member of the SNF2 family,
DECREASE IN DNA METHYLATION1 (DDM1) (Jeddeloh et al.,
1999; Brzeski and Jerzmanowski, 2003), is required for the
maintenance of heterochromatin DNA CpG and histone H3K9
methylation. DDM1 also controls the zygotic stability of parent-oforigin imprinting effects caused by mutations of the MEDEA,
FERTILIZATION-INDEPENDENT SEED2 (FIS2), and FERTILIZATION INDEPENDENT ENDOSPERM genes, which code for PcG
repressors of fertilization-independent endosperm development
(Luo et al., 2000; Gendrel et al., 2002). On the other hand, DRM1
(a member of the RAD54/ATRX SNF2-like ATPase subfamily
[Kanno et al., 2004]) is involved in the maintenance of RNA-
2455
directed non-CpG (i.e., CpNpG and CpNpN) methylation,
whereas MORPHEUS MOLECULE1 (a protein carrying part of
a SNF2-like ATPase domain [Amedeo et al., 2000]) is required for
transcriptional gene silencing.
In contrast to the multiplicity of SNF2-like ATPase proteins,
Arabidopsis has only one gene, BUSHY (BSH) (Brzeski et al.,
1999), coding for a structural and functional homolog of SNF5.
Unlike yeast and mammals, however, Arabidopsis contains as
many as four genes encoding SWI3 homologs (Sarnowski et al.,
2002). Because the BSH and ATSWI3 proteins are predicted to
form various SWI/SNF complexes with different SWI2/SNF2
subunits, mutations that affect the BSH and SWI3 genes are
expected to cause defects that are more severe than or overlapping with those that result from mutations of various SWI2/
SNF2 subunits. In fact, partial silencing of BSH in seedlings using
an antisense approach led to complex pleiotropic defects, including infertility and reduction of apical dominance (Brzeski
et al., 1999), whereas similar silencing of ATSWI3B/CHB2
resulted in dwarfism, delayed flowering, and abnormal seedling
and leaf development (Zhou et al., 2003). The multiplicity of
ATSWI3 genes and the finding that ATSWI3B interacts with FCA,
a putative RNA binding protein that regulates flowering time,
supports the hypothesis that Arabidopsis may use specialized
SWI/SNF complexes for various chromatin-based regulatory
functions (Sarnowski et al., 2002). Here, we describe the characterization of knockout mutations in all four Arabidopsis ATSWI3
genes, which indicates that, compared with yeast and animals,
the members of the Arabidopsis SWI3 family have undergone
considerable functional diversification. Mutations in ATSWI3A and
ATSWI3B cause similar blocks of embryo development at the
early globular stage. However, unlike atswi3a, the atswi3b mutations result in aberrant segregation of progeny with arrested
ovules, which suggests a possible role for ATSWI3B in imprinting.
By contrast, mutations in ATSWI3C and ATSWI3D do not prevent
embryonic development but cause characteristic alterations in
the development of vegetative and reproductive organs as well
as an early-flowering phenotype that is characterized by a reduction in the number of rosette leaves under noninductive conditions.
RESULTS
Classification of Plant SWI3 Proteins
Analysis of the SWI3 superfamily shows that plants have the
largest number of nonallelic SWI3 isoforms among multicellular
organisms (see the Plant Chromatin Database at http://chromdb.
org). Phylogenetic analysis indicates that the sequenced Arabidopsis and rice (Oryza sativa) genomes encode two distinct SWI3
subfamilies (Figure 1A; see Supplemental Table 1 online). The A/B
family is composed of two closely related branches, one with
the Arabidopsis ATSWI3A and rice Chb703 proteins, and another
with ATSWI3B and Chb702. The C/D family branches include
ATSWI3C and the related rice proteins Chb701 and Chb705 as
well as ATSWI3D with its closest rice homolog, Chb704. The
topology of the phylogeny tree predicts that the appearance of
four SWI3 subfamilies preceded the separation of monocotyledonous and dicotyledonous plant species during evolution.
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Figure 1. Phylogenetic Tree and Domain Organization of Arabidopsis SWI3-Type Proteins.
(A) A consensus maximum parsimony tree from phylogenetic analysis of SWI3-type proteins was constructed. The names of SWI3 proteins from
different species along with the accession numbers of the corresponding sequences are listed. Numbers on each branch represent the corresponding
bootstrap probability values obtained in 1000 replications.
(B) Protein domains in yeast and Arabidopsis SWI3 proteins were identified using the PFAM-LS database of global domain hidden Markov models
(Bateman et al., 1999) and the EMBOSS implementation of the HMMER package (Rice et al., 2000).
Compared with the situation in animals and humans, which have
either one (Drosophila) or two (Caenorhabditis elegans, mammals, and humans) SWI3 variants, an early duplication of plant
SWI3 families suggests a potential functional diversification
between their members. In contrast with a previous report by
Zhou et al. (2003), comparative analysis of conserved domains
using the Smith–Waterman algorithm (Smith and Waterman,
1981) as well as a search of the PFAM_LS database of global
domain hidden Markov models (Bateman et al., 1999) showed
that all characteristic SWI3 domains, including a SWIRM domain
Control of Development by SWI3 Proteins
(Swi3/Rsc8/Moira), a SANT domain, and a Leu zipper (Crosby
et al., 1999), are present in all four Arabidopsis SWI3 proteins
(Figure 1B). However, the length and sequence of interdomain
regions vary significantly among members of the A/B and C/D
subfamilies.
Interactions between Arabidopsis SWI3 Proteins in
the Yeast Two-Hybrid System
In our previous studies, we demonstrated that ATSWI3B can
form homodimers as well as heterodimers with ATSWI3A,
ATSWI3C, and BSH/SNF5 in the yeast two-hybrid system
(Sarnowski et al., 2002). In addition, we found that ATSWI3B
could recruit proteins carrying RNA binding RRM motifs, including FCA, a positive regulator of flower transition (Macknight
et al., 2002). By assaying protein interactions of other ATSWI3
family members (Figure 2A), we observed that ATSWI3A can also
form homodimers as well as heterodimers with ATSWI3C. In
addition to binding ATSWI3B, ATSWI3A also showed interaction
with BSH/SNF5 and with the C-terminal region of FCA, which
also mediates the interaction with ATSWI3B (Sarnowski et al.,
2002). ATSWI3A and ATSWI3B thus recognized similar partners
in the two-hybrid assays. However, we failed to detect an interaction between ATSWI3C and BSH. Therefore, it is possible
that ATSWI3C associates with BSH only by forming a heterodimer with either ATSWI3A or ATSWI3B. By contrast, ATSWI3A
and ATSWI3B may occur in complex with BSH as either
a homodimer or a heterodimer. ATSWI3D recognized only
ATSWI3B as a binding partner and failed to interact with BSH.
This finding suggested that ATSWI3D may recruit BSH only in
a heterodimeric complex with AtSWI3B (Figure 2B). Finally, when
tethered to DNA by fusion with the Gal4 DNA binding domain,
both ATSWI3C and ATSWI3D were capable of activating the
transcription of the lacZ reporter gene, which was not observed
with the ATSWI3A and AtSWI3B baits in yeast.
Identification of T-DNA Insertion Mutations in Genes
Encoding ATSWI3 Homologs
Arabidopsis lines carrying T-DNA insertions in the ATSWI3 genes
were identified by PCR screening of different insertion mutant
populations (Figure 3). Three atswi3a mutant alleles were found in
the SALK collection (Alonso et al., 2003). The atswi3a-1 allele
carries a single T-DNA tag (with an intact left border junction and
a deletion of 125 bp at the right border; see Methods) in the third
exon, replacing a target site deletion of 35 bp. The other mutant
alleles, atswi3a-2 and atswi3a-3, contain inverted T-DNA repeats
with left border junctions at their termini in the second and
third exons, respectively. The T-DNA insertions generated deletions of 54 and 44 bp in the atswi3a-2 and atswi3a-3 alleles,
respectively (Figure 3A). In the ATSWI3B gene, two T-DNA
tags were identified. The atswi3b-1 allele was isolated by
PCR screening of our collection (Rı́os et al., 2002), whereas
atswi3b-2 was found in the GABI-Kat flanking sequence tag
database (Rosso et al., 2003). Both atswi3b-1 and atswi3b-2 carry
inverted T-DNA repeats with left border junctions at their termini
in the third and first exons, respectively (Figure 3B). The T-DNA
integration events were accompanied by deletions of 37 and
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47 bp in atswi3b-1 and atswi3b-2, respectively. In addition, insertion of the T-DNA in the first exon added an in-frame stop codon
21 bp downstream of the ATG in atswi3b-2.
Two T-DNA insertions inactivating ATSWI3C were identified in
our collection. The atswi3c-1 allele harbors a single-copy T-DNA
insertion within a target site deletion of 42 bp in the third exon
(Figure 3C). The atswi3c-2 allele contains an inverted T-DNA
repeat with left border junctions facing the boundaries of a deletion of 31 bp in the sixth intron. Although the latter tag is located
in an intron, atswi3c-1 and atswi3c-2 confer identical mutant
phenotypes. In the atswi3d-1 and atswi3d-2 mutant alleles
derived from the SALK lines and our collection, we found inverted
T-DNA repeats with left border junctions at their termini, which
defined the break points of deletions of 24 and 8 bp in the third
and fifth exons of the ATSWI3D gene, respectively (Figure 3D). All
T-DNA–tagged mutant lines identified by PCR screening were
backcrossed with the wild type, verified by DNA gel blot
hybridization and also by segregation analysis of the T-DNA–
encoded antibiotic resistance genes (except for the SALK lines,
which all carried silenced selectable markers; see Methods).
Mutations in ATSWI3A Affect Early Embryo Development
In the segregating SALK mutant lines, we failed to identify
homozygous atswi3a mutants. Hence, for each T-DNA insertion
allele, we examined the progeny of 30 PCR-genotyped atswi3a/þ
plants after self-pollination. Young fruits of all atswi3a/þ lines
contained ;25% white translucent seeds that degenerated into
collapsed, brown aborted seeds during maturation (Figure 4A;
see Methods). This observation suggested that independent of
the position of T-DNA tags in the ATSWI3A gene, all three
insertion mutant alleles resulted in a similar, recessive embryolethal phenotype. To confirm this conclusion, we compared the
developmental status of embryos in green wild-type seeds and
white aborted seeds 4 d after fertilization (Figure 4B). Compared
with wild-type seeds, which carried embryos in the heart stage,
all white seeds showed delayed development and carried
embryos arrested in the late globular stage. Although cell
divisions appeared to occur normally in the suspensor, the embryo proper displayed aberrant globular shape and degeneration, indicating that ATSWI3A function is essential for proper
early embryo development.
Mutations of ATSWI3B Result in Early Embryo Lethality and
Gametophytic Defects Causing Distorted Segregation
By PCR screening, we also failed to identify homozygous atswi3b
mutants. Therefore, we examined the segregation of hygromycin
and sulfadiazine resistance markers of T-DNA tags of atswi3b-1
and atswi3b-2 alleles, respectively. All M2 lines that carried
single T-DNA tags showed a segregation ratio of 2:1 (resistant:
sensitive) progeny (see Methods). PCR genotyping of 50 M3
plants confirmed that the resistant lines were heterozygous for
the mutant atswi3b alleles. Thus, the segregation data suggested
that the atswi3b mutations caused recessive embryo lethality.
However, inspection of young siliques of atswi3b-1/þ and
atswi3b-2/þ plants revealed an unusual segregation of three
distinct phenotypes (Figures 5A and 5B). In addition to green
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Figure 2. Yeast Two-Hybrid Assays with Arabidopsis SWI3 Proteins.
(A) b-Galactosidase filter lift assays of pair-wise interactions between
ATSWI3A and BSH, ATSWI3C and ATSWI3A, ATSWI3A and FCA,
ATSWI3C and BSH, ATSWI3D and ATSWI3A, and ATSWI3B and BSH.
Blue indicates protein interactions. Control self-activation assays are
shown at left in yeast strains carrying ATSWI3A, ATSWI3B, ATSWI3C,
ATSWI3D, and FCA bait (BD, fusion with the Gal4 DNA binding domain)
or prey (AD, fusion with the Gal4 activation domain) constructs in
combination with empty bait or prey vectors, respectively. Note that
the ATSWI3C-BD and ATSWI3D-BD baits show self-activation in yeast.
Small geometric shapes indicate the domain organization of FCA. The
FCA-AD prey encodes full-length FCA, whereas FCA-PhD-AD codes for
a C-terminally truncated form of FCA lacking the ATSWI3B-interacting
region.
(B) Model for combinatorial interactions between ATSWI3, BSH, and
wild-type seeds, representing 40 to 41% of progeny, the fruits
contained 48 to 49% arrested ovules and 10 to 11% white
aborting seeds, which turned brown and collapsed within 6 to
7 d after fertilization (Table 1). Inspection of embryos 4 d after
pollination using Nomarski optics showed that compared with
wild-type seeds, which carried late-heart-stage embryos, the
white aborting seeds were delayed in development and contained aberrant globular embryos (Figure 5C). A more detailed
cytological analysis showed that in white seeds, the endosperm
was not cellularized, which is a characteristic trait for early stages
of seed development. The embryos displayed abnormal cell
shapes and division patterns in the embryo proper and suspensor and remained arrested in the early globular stage (Figure 5D).
To characterize the defect leading to the formation of arrested
ovules, we examined the process of megasporogenesis before
fertilization in atswi3b/þ plants. Although apparently normal
megaspores were detected in all ovules after meiosis, ;50%
of ovules failed to form embryo sacs; thus, the megaspores did
not undergo further divisions but remained arrested in a central
position (Figure 5D). As the number of wild-type plus white seeds
and that of arrested ovules yielded a segregation ratio of ;1:1,
whereas the T-DNA–tagged atswi3b and wild-type alleles segregated at a ratio of 2:1, the arrested ovule phenotype appeared
to be independent of the atswi3b mutations. Therefore, we
conclude that the recessive atswi3b mutations resulted in the
early embryo lethality seen in white aborting seeds, although the
ratio between wild-type and white seeds (;3.6:1) deviated
somewhat from the expected 3:1 ratio. Thus, the phenotypes
of the atswi3a and atswi3b mutations appeared to be very similar,
although the atswi3b mutations led to earlier arrest of globular
embryos and aberrant cell division patterns in the suspensor.
To study the inheritance of the arrested ovule phenotype, we
performed recurrent reciprocal crosses between heterozygous
atswi3b/þ and wild-type plants (Table 1). When using the wild
type as the male pollen donor, we observed an ;1:1 segregation
of green seeds and arrested ovules in the hybrid fruits. This
segregation ratio suggested female gametophytic lethality (i.e.,
predicting no female transmission of the mutant atswi3b alleles).
However, germination of wild-type F1 seeds found in the hybrid
siliques revealed a 1:1 segregation of T-DNA–tagged atswi3b
and wild-type alleles, clearly excluding this possibility. Inspection of 5060 ovules in the F2 progeny of self-pollinated atswi3b/þ
F1 plants revealed a segregation of 37.4% 6 0.9% green wildtype seeds and 11.5% 6 0.4% white mutant seeds as well as
51.1% 6 0.9% arrested ovules. In hybrid siliques of the reciprocal cross obtained by pollination of the wild type with either
atswi3b-1/þ or atswi3b-2/þ pollen donors, we observed 82 to
87% wild-type seeds, but also a consistent appearance of ;12
to 17% unfertilized ovules. Viable progeny from these reciprocal
crosses also showed a 1:1 segregation of T-DNA–tagged mutant
and wild-type alleles, indicating successful male transmission of
atswi3b alleles. Upon self-pollination of reciprocal atswi3b/þ F1
hybrids, we scored 4252 F2 progeny, which consisted of 36.5%
FCA proteins based on the interaction screens in (A) and earlier results
obtained by Sarnowski et al. (2002). Circles indicate the homodimerization capability of ATSWI3A and ATSWI3B.
Control of Development by SWI3 Proteins
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Figure 3. Schemes of the Positions, Orientation, and Organization of T-DNA Insertions in the ATSWI3A, ATSWI3B, ATSWI3C, and ATSWI3D Genes.
The positions of exons (gray boxes with numbers) and introns are indicated. The boundaries of T-DNA inserts (white boxes) marked as LB or RB refer to
the junction sequences described in Methods. PCR primers used to identify genotypes of insertion mutants are labeled by arrowheads.
(A) ATSWI3A.
(B) ATSWI3B.
(C) ATSWI3C.
(D) ATSWI3D.
6 0.8% wild-type seeds and 11.5% 6 0.6% white mutant seeds
as well as 52.0% 6 0.7% arrested ovules. After two backcrosses
with the wild type, the heterozygous atswi3b-1/þ and atswi3b-2/þ
families showed similar segregation ratios of arrested ovule and
aborting seed phenotypes as the original heterozygous M2 mutant
families, indicating stable inheritance of both traits.
The appearance of unfertilized ovules in wild-type carpels
pollinated by atswi3b/þ males suggested partial male sterility,
which implied that the atswi3b mutations could also influence
male gametogenesis in addition to affecting macrosporogenesis. Examination of microsporogenesis in atswi3b/þ plants
showed that the pollen mother cells could undergo meiosis,
producing callose-separated tetrads and later fully separated
vacuolated microspores (Figure 5E). However, only approximately half of the microspores (245 of 472 examined) entered
into further division, whereas the rest remained vacuolated and
exhibited senescence (i.e., lipid deposition, organelle degradation; data not shown) or collapsed. Thus, the observed severe
defects in microsporogenesis provided a plausible explanation
for the occurrence of unfertilized ovules in fruits derived from the
cross of wild-type females with atswi3b/þ males. As the atswi3b
mutations were transmitted by the female, we could also exclude
the possibility that potential imprinting of the wild-type ATSWI3B
male allele caused a defect immediately after fertilization or later
during embryo development. However, the strikingly different
results of reciprocal crosses, which contradicted Mendel’s first
law on the uniformity of F1 hybrids, suggested that the potential
haploinsufficiency of ATSWI3B might lead to the imprinting of
some genes essential for mitotic divisions of macrospores or
microspores, or both. The fact that the atswi3b alleles showed
similar male and female transmission suggested that, if there
were any imprinted genes, they were probably unlinked at the
ATSWI3B locus. We examined 196 M3 offspring of a selfpollinated atswi3b/þ plant. From these, 65 AtSWI3B/AtSWI3B
lines produced only wild-type seeds, 126 atswi3b/þ lines segregated wild-type and white seeds together with arrested ovules,
4 atswi3b/þ lines had only wild-type and white aborted seeds,
and 1 ATSWI3B/ATSWI3B line segregated only arrested ovules.
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Figure 4. T-DNA Insertions in the ATSWI3A Gene Cause Embryo
Lethality.
(A) Open siliques of a wild-type control plant and an atswi3a-1/þ plant
showing segregation of green wild-type and white aborting seeds
(arrow).
(B) Nomarski images of the same age wild-type and mutant (atswi3a-1/
atswi3a-1) seeds, and enlarged sections showing a normal heart-stage
embryo in the wild type and an aberrant globular-stage embryo in the
mutant. Bar ¼ 100 mm.
After crossing the last line with a wild-type male, the fruits of F1
hybrids contained 30 to 40% aborted ovules in addition to wildtype seeds. Thus, it appeared that the locus (or loci) conferring
the arrested-ovule phenotype was genetically separable from the
atswi3b locus (Figure 5F). This observation, of course, raised the
possibility that the aborted-ovule phenotype was caused by an
independent mutation and not by an atswi3b-dependent imprinting effect. However, the observed aberrant inheritance of
the arrested-ovule phenotype, which did not fit Mendelian
predictions, its transmission into the F2 progeny obtained by
recurrent reciprocal backcrosses with the wild type, and its
occurrence in two independent atswi3b mutant lines suggested
instead that the arrested-ovule phenotype is an epigenetic trait,
which may be further targeted by mapping experiments to verify
a potential role of ATSWI3B in imprinting.
Inactivation of ATSWI3C Causes Alterations in Leaf, Root,
and Flower Development
In contrast to atswi3a and atswi3b, PCR screening identified
viable plants homozygous for the atswi3c-1 and atswi3c-2
mutations. RT-PCR analysis revealed an absence of wild-type
ATSWI3C transcript in the homozygous mutants, indicating that
both atswi3c alleles represented null mutations (Figure 6A).
Plants carrying the atswi3c-1 and atswi3c-2 alleles showed
identical phenotypic alterations, including delayed development,
semidwarf growth habit, and formation of aberrant rosettes
(Figure 6B). The rosette and cauline leaves were severely twisted
along the proximodistal (base-to-tip) axis, which led to a downward curvature of the lateral edges toward the abaxial leaf
surface. Cross sections of leaves showed an increased protrusion of mesophyll cell files around the vascular bundles (Figure
6C). This phenotype resembled those caused by the CURLY
LEAF (Polycomb repressor of AGAMOUS) and pie (ISWI-type
SWI2/SNF2 ATPase) mutations (Kim et al., 1998; Noh and
Amasino, 2003; Katz et al., 2004) and by silencing of the SWI2/
SNF2-like gene BRAHMA (Farrona et al., 2004). The root system
of homozygous atswi3c seedlings was greatly reduced, because
the elongation of primary roots was inhibited and the seedlings
developed several side roots and secondary root branches
(Figure 6D).
When grown in soil under inductive long-day (LD) conditions
(16 h light/8 h dark), the atswi3c mutants flowered 24 to 25 d after
planting, with a mean leaf number between 9 and 10, whereas
wild-type plants segregating in the same pots flowered at days
20 and 21, with a mean leaf number of 11 (Figure 7A). Under
noninductive short-day (SD) conditions (8 h light/16 h dark), the
atswi3c mutants flowered between 65 and 75 d after planting,
with a mean leaf number of 27, whereas the wild type flowered
between 58 and 67 d after planting, with a mean leaf number of
54 (Figure 7B). As in other phenotypic traits, plants carrying the
atswi3c-1 and atswi3c-2 alleles showed no difference in flowering time behavior. Based on leaf number, both atswi3c mutants
were early flowering during SD conditions and slightly early
during LD conditions. In addition to reduced leaf number, both
atswi3c mutants had reduced numbers of cauline leaves on the
primary inflorescence stem (mean three to four versus eight in the
wild type; Figure 7C) and fewer secondary inflorescences (mean
two versus eight in the wild type; Figure 7D).
To monitor the developmental regulation of flowering time
integrator and floral homeotic genes (for recent reviews, see
Ferrario et al., 2004; He and Amasino, 2004) during SD conditions, a series of semiquantitative RT-PCR assays was performed. RNA samples were prepared from whole atswi3c-1
mutant and wild-type seedlings, excluding roots, at days 15, 31,
and 41 after planting, as well as from tips of emerging inflorescences on the first day of flowering (i.e., day 66, overlapping
between the periods of onset of flowering in the atswi3c mutants
and the wild type). The RT-PCR assays with all RNA samples
were performed using three different PCR cycle numbers and
ACTIN2 as an internal control. Although FT and FLC transcript
levels were 1.8- and 2-fold higher in mutant than in wild-type
seedlings at days 31 and 41, respectively, no dramatic change in
the regulation of flowering time integrator genes was observed in
the atswi3c-1 mutant during SD conditions (Figure 8). RT-PCR
analyses with plants grown under LD conditions yielded similar
data, except that compared with the wild type, FT transcript
levels were reduced by 30 to 50% in samples collected from
atswi3c-1 seedlings 15 d after planting and from emerging
inflorescence tips on the first day of flowering (data not shown).
In contrast to the wild type, no APETALA3 (AP3) transcript was
detected in vegetative tissues of the atswi3c-1 mutant, whereas
PISTILLATA (PI) transcript levels showed a reduction before
flowering. In inflorescence tips carrying the differentiating floral
meristems, transcript levels of the floral homeotic genes AP1,
Control of Development by SWI3 Proteins
2461
Figure 5. Mutations of the ATSWI3B Gene Cause Embryo Lethality and Non-Mendelian Segregation of Arrested Ovules Showing a Defect in
Megasporogenesis.
(A) Open siliques of wild-type control and atswi3b-1/þ mutant plants. A red arrow marks an arrested embryo resulting from maternal imprinting of the
atswi3b-1 mutation. Bar ¼ 0.5 mm. The bottom panel shows a scanning electron microscopic image of wild-type seeds and an arrested ovule. Bar ¼
100 mm.
(B) Longitudinal cross section of an atswi3b-1/þ silique 4 d after pollination showing the structures of aborting (top row, first embryo from left) and wildtype (top row, second embryo from left) seeds and arrested ovules (bottom row, red arrow). Bar ¼ 100 mm.
(C) Nomarski images of a heart-stage embryo in the wild type and an aberrant globular embryo (black arrow) in atswi3b-1/atswi3b-1 mutant seeds.
Bar ¼ 100 mm.
(D) Top, the cross section at left shows the embryo sac of an unfertilized wild-type ovule with a typical arrangement of synergids and egg cell at the
micropylar pole and antipodal cells at the chalazal pole. The cross section at right shows an arrested ovule, which contains no embryo sac and displays
a centrally entrapped vacuolated macrospore. Bottom, cross sections of globular embryos from a wild-type seed (left) and a white aborting seed (right).
In the white aborting atswi3b-2 seed, the defective globular embryo shows aberrant cell divisions in the suspensor, unusual shape of the hypophysis
cell, and degeneration of internal cells in the embryo proper. Bars ¼ 50 mm.
(E) Stamen cross sections showing the process of microsporogenesis in the atswi3b-2/þ mutant. Top, images from left to right show the normal division
of a pollen mother cell, formation of the tetrads, resolution of callose layers connecting the tetrads, and release of the tetrads. Bottom, after separation
of the tetrads, ;50% of microspores fail to divide and undergo vacuolization, senescence, and degeneration. The cross section of an anther shows
lobules containing a mixture of wild-type and degenerated spores. Bar ¼ 50 mm.
(F) The white aborting seed phenotype linked to the atswi3b-1 locus and the arrested ovule phenotype, resulting from atswi3b-1–mediated imprinting,
segregate as unlinked traits.
AP3, and PI were significantly lower in the atswi3c-1 mutant than
in the wild type under short days (Figure 8). In addition to these
genes controlling the development of sepals, petals, and stamens, AP2 transcript levels also showed a reduction in inflorescence tips of LD-grown mutant plants (data not shown). In
association with these transcriptional changes, the atswi3c
mutants displayed a 40 to 50% size reduction of flowers, which
was especially severe under short days and low humidity and
more pronounced in basipetal flowers, as described in
BRAHMA-silenced plants by Farrona et al. (2004). However,
the size of mature flowers under LD conditions was comparable
to that of the wild type.
2462
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Table 1. Seed Composition of F1 Siliques Obtained by Self-Pollination and Crosses between atswi3b/1 and Wild-Type Plants
Female 3 Male
Wild-Type
Seeds (%)
Wild-Type
Segregation (R:Sa)
White
Aborted Seeds (%)
Arrested
Ovules (%)
No.
atswi3b-1/þ 3 atswi3b-1/þ
atswi3b-1/þ 3 wild type
Wild type 3 atswi3b-1/þ
atswi3b-2/þ 3 atswi3b-2/þ
atswi3b-2/þ 3 wild type
Wild type 3 atswi3b-2/þ
Wild type 3 wild type
atswi3b-1/þ (second backcross)
atswi3b-2/þ (second backcross)
41
46
87
40
45
80
98
39
38
2:1
1:1
1:1
2:1
1:1
1:1
–
2:1
2:1
11
<1
<1
11
<1
<1
<1
11
11
48
53
12
49
55
20
1
50
51
2000
494
2000
2000
752
1668
1301
1047
1024
Both atswi3b mutants were backcrossed twice with the wild type.
R, resistant; S, sensitive.
a
More than 80% of mutant flowers contained a fused pair of
stamens with degenerated twin anthers. The anthers displayed
greatly reduced dehiscences and frequent disjunction between
the pair of locules. Occasionally, the anthers were replaced by
sepalloid tissues. In addition, approximately one-third of the
flowers contained one or two staminoid filaments or arrested
stamen primordia (corresponding to a reduction in the number of
stamens; Figures 6E and 6F). The atswi3c mutants showed
greatly reduced fertility. Although the pollen grains appeared to
be normal, during maturation the majority of them remained
entrapped and degenerated in the anthers. The siliques contained only a few, often degenerating seeds. Even after manual
fertilization with wild-type pollen, fruit development remained
abnormal and few viable seeds were obtained. The atswi3c
mutants showed distorted segregation of the T-DNA–encoded
hygromycin resistance marker. Self-fertilization of the atswi3c-1
mutant, for example, yielded a 2.5:0.4:1.1 segregation of resistant wild-type, mutant, and sensitive offspring, indicating
a reduction in the homozygous mutant class (homogeneity
x2 ¼ 0.378, P ¼ 107). As no aborting seeds were observed in
the siliques of self-pollinated atswi3c/þ plants, the segregation
data suggested a possible decrease in either male transmission or
the viability of atswi3c gametes. However, because homozygous
mutant seeds could be recovered, the development of mutant
embryos in atswi3c/þ plants appeared to be unaffected. By
contrast, in homozygous atswi3c mutants, the recovery of seed
progeny was dramatically compromised, which indicated that
the atswi3c mutations caused pleiotropic defects in sporophytic
organ development, but unlike atswi3a and atswi3b, they did not
result in embryo lethality.
Mutations in ATSWI3D Affect the Number and Development
of Leaves and Flower Organs
Like the atswi3c mutants, plants homozygous for the atswi3d-1
and atswi3d-2 mutations were viable and could readily be
identified by PCR screening. RT-PCR analysis confirmed a lack
of wild-type ATSWI3D RNA in the homozygous lines (Figure 9A),
indicating that both atswi3d alleles represented null mutations.
Unlike the atswi3c mutants, homozygous mutant seedlings
could not be distinguished from wild-type seedlings soon after
germination, as they showed normal leaf shape and root elongation. However, plants grown in soil for 15 to 20 d displayed
slower development and dwarfism (Figures 9B and 9C). Downward curling of the tips of rosette leaves provided a useful trait for
the identification of homozygous mutants (Figure 9C).
The flowering time behavior of both atswi3d mutants was
tested under exactly the same conditions described above for
the atswi3c mutants. Both atswi3d mutants flowered under long
days, with a mean leaf number of 11, similar to the wild type but 4
to 5 d later (Figure 7A). Under noninductive SD conditions, the
atswi3d mutants flowered with a mean leaf number of 34 (54 in
the wild type; Figure 7B) between days 60 and 75 after planting
(the wild type flowered between days 58 and 67). The atswi3d
mutations also reduced the number of cauline leaves on the
primary inflorescence (mean between six and seven; eight in the
wild type) and the number of secondary inflorescences (mean
between six and seven; eight in the wild type) (Figures 7C and
7D). Based on leaf number, the atswi3d mutants were classified
as early flowering during short days but showed a less severe
flowering time phenotype compared with the atswi3c mutants.
The developmental regulation of flowering time integrator and
floral homeotic genes was monitored in the atswi3d-1 mutant by
RT-PCR assays as described above for atswi3c-1. Compared
with the wild type, the transcript levels of FT and FLC were 5- and
2.5-fold higher at days 31 and 41, respectively, after planting in
atswi3d-1 seedlings under SD conditions. Although FT and FLC
RNA levels also showed some differences at day 15 and later in
mutant and wild-type plants grown under LD conditions (data not
shown), the changes detected by RT-PCR did not correlate with
the observed early-flowering phenotype and with the comparable transcription of CO and SOC1 genes in mutant and wild-type
plants grown under SD and LD conditions. Intriguingly, transcript
levels of AP3 and PI were threefold to sevenfold greater, whereas
no AG RNA was detected at days 31 and 41 after planting in
atswi3d-1 seedlings compared with the wild type. However, in the
tips of inflorescences, AG appeared to be transcribed normally,
whereas transcript levels of AP1, AP3, PI, and UFO were reduced
twofold or more compared with the wild type. As seen in the
atswi3c-1 mutant, in addition to these genes, AP2 also showed
reduced transcript levels in the atswi3d-1 mutant under LD
conditions (data not shown).
Control of Development by SWI3 Proteins
2463
Figure 6. Developmental Defects Caused by the atswi3c Mutations.
(A) RT-PCR assay of the wild type and homozygous atswi3c-1 and atswi3c-2 mutant lines with ATSWI3C and control ACTIN2 primers (see Methods).
The absence of PCR product with the gene-specific primer indicates that the atswi3c alleles correspond to null mutations. The size marker is a 1-kb
DNA ladder.
(B) Left, rosette phenotypes of 3-week-old atswi3c-1/atswi3c-1 mutant and wild-type plants. Bar ¼ 1 cm. Right, size comparison of 91-d-old wild-type
and atswi3c-1 (left pot) and wild-type and atswi3c-2 (right pot) plants grown under short days. Bar ¼ 5 cm.
(C) Cauline leaves of homozygous atswi3c-1 and wild-type plants are shown at top left. Bar ¼ 1 cm. Cross sections of atswi3c-1 mutant (top right) and
wild-type (bottom) leaves indicate the enlargement of mesophyll cell files around the vascular bundles in the mutant. Bars ¼ 500 mm.
(D) Left, compared with the wild type, the elongation of the primary root is inhibited in atswi3c-1 (top) and atswi3c-2 (bottom) plants, which display
enhanced side root initiation and root branching. Bars ¼ 1 cm. Top right, comparison of mutant and wild-type siliques indicates that the atswi3c-1
mutation inhibits normal fruit development and elongation. Bar ¼ 1 mm.
(E) Left, in whorl C, flowers of the atswi3c-1 mutant contain at least one fused twin stamen and one or two additional staminoid filaments (arrow). Middle
and right, compared with the wild type, stamens in atswi3c-1 flowers occasionally carry petalloid tissues in place of anthers (arrow). Bars ¼ 0.25 mm.
(F) Left and middle, scanning electron microscope images of twin anthers (left) and an arrested stamen primordium (middle) from atswi3c-1 flowers.
Right, cross section of a mutant flower showing four petals and sepals surrounding three normal and two aberrant stamens and the carpel in the center.
Bars ¼ 100 mm.
2464
The Plant Cell
Figure 7. Number of Rosette Leaves under Long and Short Days, and Number of Cauline Leaves and Secondary Inflorescence Stems under Short
Days, in the atswi3c and atswi3d Mutants.
(A) To score the number of rosette leaves under long days, segregating families of atswi3c-1 (n ¼ 104), atswi3c-2 (n ¼ 66), atswi3d-1 (n ¼ 100), and
atswi3d-2 (n ¼ 329) were germinated in soil. The data were combined for presentation of both atswi3c and both atswi3d alleles, as pairwise analysis
revealed identical flowering time behavior for both.
(B) Leaf number under the SD condition was scored in 61 atswi3c, 108 atswi3d, and 176 wild-type plants.
(C) The number of cauline leaves was counted on primary inflorescence stems of 29 atswi3c, 30 atswi3d, and 45 wild-type plants.
(D) The number of secondary inflorescences was scored using 30 atswi3c, 29 atswi3d, and 65 wild-type plants.
A general downregulation of the transcription of floral homeotic genes, except LFY, correlated with severe developmental
defects of atswi3d flowers. The sizes of sepals, petals, and
stamens of mature mutant flowers were 50 to 60% of wild-type
sizes (Figure 9D). The first two whorls of mutant flowers contained five sepals and a variable number of partially developed
petals, some of which appeared as aberrant sepal-like filaments.
The atswi3d mutants occasionally produced fused twin stamens,
like the atswi3c mutants, but with a lower frequency (Figure 9E).
The stamens carried small degenerated anthers that failed to
produce pollen. The carpels were distorted and highly degenerated; their lower section appeared as an extension of the
inflorescence stem. Because of a complete lack of fertilization,
the homozygous mutant plants produced no seeds and did not
carry normally developed siliques. Despite some similarities, the
developmental defects caused by the atswi3d mutations thus
appeared to be much more severe than and distinct from those
observed in the atswi3c mutants.
DISCUSSION
The SWI/SNF ABC: Arabidopsis SWI3 Family Members
and Their Potential Interactions
Compared with yeast, Caenorhabditis, and mammals, the number of genes encoding SWI3 homologs is duplicated from two to
four in Arabidopsis. Although the genome annotation is still
incomplete, rice probably contains even more, at least five SWI3
genes. The Arabidopsis and rice SWI3 proteins can be classified
in two families, the branching of which probably preceded the
separation of monocotyledonous and dicotyledonous species.
ATSWI3A and ATSWI3B are related to yeast Rsc8p both in size
(57, 52, and 63 kD, respectively) and domain organization. Both
ATSWI3A and ATSWI3B carry characteristic SWIRM (Swi3/
Rsc8/Moira), Leu zipper, and SANT domains, the latter with
predicted DNA binding activity to the Myb binding consensus
AAC(G/T)G. Members of the second family, ATSWI3C and
Control of Development by SWI3 Proteins
2465
Figure 8. RT-PCR Analysis of Transcript Levels of Flowering Time Integrator and Floral Homeotic Genes during Development of the Wild Type, atswi3c-1,
and atswi3d-1 during SD Conditions.
Samples were taken at 31 and 41 d after planting (DAP) by collecting whole seedlings, except for roots, whereas samples collected at day 66
correspond to inflorescence tips on the first day of flowering. Numbers under mutant names indicate (from left to right) signal intensities compared with
RT-PCR determined with control wild-type RNA samples collected at days 31, 41, and 66. Black framed images show RT-PCR products obtained with
22 cycles of PCR, whereas unframed images correspond to cDNA products amplified by 30 cycles.
ATSWI3D, more closely resemble yeast Swi3p and Drosophila
Moira, as they are larger (88 and 108 kD), but they nonetheless
share all conserved domains with ATSWI3A and ATSWI3B.
ATSWI3D is the only member of the ATSWI3 family that contains
an additional ZnF-ZZ (CxxCxxxC) domain. The functional significance of SWI3 domains is indicated by site-specific mutagenesis studies in yeast and Drosophila. Deletion of the SANT
domain results in defective SWI/SNF complexes in yeast (Boyer
et al., 2002), whereas the removal of the C-terminal Leu zipper
domain decreases the ability of Moira to self-associate in vitro
(Crosby et al., 1999).
In support of sequence-based classification, members of the
A/B and C/D ATSWI3 families show different protein interaction
properties. ATSWI3A and ATSWI3B are both capable of homodimerization and can form heterodimers with each other. Both
ATSWI3A and ATSWI3B can bind ATSWI3C as well as BSH/
SNF5 and the flowering regulator FCA protein in yeast two-hybrid
assays. By contrast, ATSWI3C and ATSWI3D fail to interact with
BSH/SNF5, and ATSWI3D can only bind ATSWI3B. This suggests that ATSWI3D can only recruit BSH/SNF5 through
ATSWI3B, whereas ATSWI3C may be tethered to BSH/SNF5
through either ATSWI3A or ATSWI3B. The SNF5 subunit, which
is known to coordinate the assembly and nucleosome-remodeling
activities of SWI/SNF complexes (Geng et al., 2001), occurs as
a single isoform in all species analyzed, including Arabidopsis. Our
data suggest that the ATSWI3 subunits could interact with BSH/
SNF5 in at least six (AA, BB, AB, AC, BC, and BD) different combinations and form core SWI/SNF complexes with one of the
two known Arabidopsis SWP73 homologs (Brzeski et al., 2003)
and one specific SNF2-type ATPase subunit.
In the absence of biochemical data, it is impossible to predict
which of the 42 potential SNF2-like ATPase homologs will occur
in SWI/SNF complexes with BSH/SNF5 and the ATSWI3 proteins. The ATPases found in SWI/SNF complexes of other
species all carry characteristic SNF2_N (i.e., a variant of the
typical DEXD/H domain) and bromodomain motifs. The bromodomain is a conserved C-terminal motif of ;110 amino acids
that is implicated in the recognition of acetylated Lys residues of
core histone tails (Hudson et al., 2000). In Arabidopsis, BRAHMA
is the only SNF2/BRAHMA homolog that carries a C-terminal
bromodomain, which may enable the SWI/SNF complexes to
stimulate the activation of transcription in synergy with histone
acetyltranferases.
Recently, BRAHMA was found to interact with ATSWI3C in
the two-hybrid system (Farrona et al., 2004). As a canonical
ATPase subunit, BRAHMA may occur in SWI/SNF complexes
carrying ATSWI3C bound to either ATSWI3A or ATSWI3B and
BSH. Silencing of BRAHMA by an RNA interference approach
was found to cause an overall size reduction of vegetative and
reproductive organs, resulting in curly leaves, short sepals and
stamens, reduced fertility, and homeotic transformations between whorls B and C. Downregulation of BRAHMA resulted in
2466
The Plant Cell
Figure 9. Developmental Alterations Caused by the atswi3d Mutations.
(A) RT-PCR assay of the wild type and homozygous atswi3d-1 and atswi3d-2 mutant lines with ATSWI3D and control ACTIN2 primers (see Methods).
The size marker is a 1-kb DNA ladder.
(B) Size comparison of wild-type and dwarf atswi3d-1 (left pot) and wild-type and atswi3d-2 (right pot) plants grown for 91 d under SD conditions.
Bar ¼ 5 cm.
(C) Rosettes of atswi3d-1 mutant and wild-type plants grown for 21 d in soil. Note the downward bending of mutant rosette leaves. Bars ¼ 1 cm.
(D) Flowers of homozygous atswi3d-1 and wild-type plants (see description in text). Bar ¼ 0.5 mm.
(E) Scanning electron microscopic images of aberrant carpel development and occasional occurrence of fused twin stamens detected in atswi3d-1
mutant flowers. Bars ¼ 200 mm.
precocious flowering under both inductive and noninductive
conditions and upregulated the transcript levels of CO, FT, and
SOC1 (Farrona et al., 2004). Our studies show that T-DNA
knockout mutations of ATSWI3C cause similar morphological
alterations to BRAHMA silencing. In addition, the atswi3c null
mutants display reduced numbers of rosette leaves (i.e., ;50%
of wild type), especially under SD conditions, and can thus be
classified as early-flowering mutants, although they actually
flower 4 to 5 d later than the wild type under inductive long days
and ;8 to 10 d later during short days. In the experiments
described by Farrona et al. (2004), plants expressing BRAHMA
RNA interference flowered after 28 6 3.6 d during short days
(10 h light/14 h dark), whereas wild-type plants bolted after 59 6
3.4 d. Farrona et al. (2004) compared RNA samples prepared
from whole seedlings collected at days 10, 13, and 16 from
silenced and wild-type plants and detected significantly higher
levels of CO, FT, and SOC1 transcript in plants carrying the
BRAHMA RNA interference construct. In our experiments, wildtype plants flowered between days 58 and 67, and samples
were collected at days 15, 31, 41, and 66 after planting
However, in samples harvested at day 15, only extremely low
CO, SOC1, and FT transcript levels were found in both wild-type
and atswi3c (and atswi3d ) plants, which could only be detected
with a large number of PCR cycles. Therefore, we relied instead
on a comparison of transcript levels at later time points of
development (Figure 7). Our results suggest that the earlyflowering phenotype of atswi3c (and atswi3d) mutants did not
result from a significant upregulation of genes acting in the
photoperiod-sensitive flowering pathway. Nonetheless, more
rigorous analysis should address this question (e.g., by monitoring the activity of FT, SOC1, and CO genes using promoterluciferase fusions in the mutants throughout the full time period
Control of Development by SWI3 Proteins
between planting and bolting). In addition, testing potential
interactions with the autonomous and vernalization-dependent
flowering pathways (for review, see He and Amasino, 2004;
Henderson and Dean, 2004) should reveal whether the reduced
leaf number of atswi3c (and atswi3d) mutants simply reflects
defective sporophytic development or is a consequence of the
altered expression of genes involved in the regulation of flowering time (Boss et al., 2004). However, it is also conceivable that
in the atswi3c mutants, BRAHMA, defined as a repressor of CO
(and thereby FT and SOC1), remains capable of delaying
flowering. This suggests that BRAHMA may either act as a repressor independent of SWI/SNF complexes or occur in alternative SWI/SNF complexes together with BSH and ATSWI3A or
ATSWI3B (or possibly with ATSWI3D). Further studies should
clarify whether BRAHMA can interact with other members of the
ATSWI3 family and whether FCA (a negative regulator of FLC)
and CO (a positive regulator of photoperiod-dependent flowering) would mediate the opposite changes in flowering time
caused by the atswi3c mutations and the silencing of BRAHMA.
The interaction of FCA with ATSWI3A and ATSWI3B also
suggests that these SWI/SNF core subunits could be targeted by
FCA to its partner FY, which is implicated in the control of splicing
and 39 polyadenylation of the FCA transcripts (for review, see
Simpson et al., 2004). A recent finding indicates that the human
SNF2 ATPase hLodestar/huF2 can interact with CDC5L, a key
pre-mRNA splicing factor (Leonard et al., 2003). This suggests
that it will be necessary to examine whether the atswi3c mutations affect the splicing and polyadenylation of FCA pre-mRNA.
Through the bromodomains of BRAHMA, the ATSWI3C-containing SWI/SNF complexes may be targeted to acetylated
histone cores in transcriptionally active chromatin. The fact that
BRAHMA silencing results in precocious flowering suggests that
the BRAHMA SWI/SNF complex could act as a repressor by
recruiting histone deacetylases to the active chromatin. In fact,
mutations that cause late flowering by inactivating the FLOWERING LOCUS D (FLD) gene in the autonomous flowering pathway
were recently shown to result in the hyperacetylation of histone
H4 in FLC chromatin. FLD encodes a protein showing a close
relationship to a subunit of human HISTONE DEACETYLASE1/2
complexes (He et al., 2003). A second gene in the autonomous
flowering pathway, FVE, encodes an Rb binding protein, MSI4,
that is also required for the downregulation of FLC via histone H3
deacetylation (Ausin et al., 2004). Therefore, it will be important to
examine how the atswi3c mutations affect the histone acetylation status of ATSWI3C-regulated genes.
Inactivation of ATSWI3C and ATSWI3D Causes Distinct
Defects in the Development of Vegetative
and Reproductive Organs
In addition to reducing leaf number at flowering, mutations in
ATSWI3C cause characteristic defects in vegetative organ development, which include the formation of curly leaves and the
inhibition of primary root elongation, leading to enhanced root
branching. The atswi3c mutations also result in a high degree of
male and female sterility as well as the appearance of fused twin
stamens and petalloid bracts in whorl B. These developmental
alterations are accompanied by a significant decrease in AP1, PI,
2467
and AP3 transcript levels in the inflorescence tips of atswi3c
mutants grown under both SD and LD conditions. The phenotype
of atswi3d mutants clearly differs from that of atswi3c mutants,
although both mutations affect flower development and leaf
number at flowering. The atswi3d mutations do not result in the
typical curly-leaf phenotype and inhibition of root elongation, but
they cause severe developmental defects in all floral whorls. The
mutant flowers contain five sepals in whorl A and defective organs
in whorl B, including shorter and degenerated petals and petalloid
filaments. Because of severe deficiencies in anther and carpel
development, the homozygous mutants are sterile. The atswi3d
mutations result in a reduction of expression of AP1, AP3, PI, and
UFO genes in inflorescence tips, but they also alter the expression
levels of AP3, PI, and AG in sporophytic tissues before flowering
(Figure 7). Thus, in addition to ATSWI3C, ATSWI3D also appears to
be an important regulator of floral organ identity and development.
The pleiotropic phenotype of atswi3d mutations shows no resemblance to known mutations of SNF2-like ATPases. Thus,
better understanding of ATSWI3D function requires further genetic
dissection of the interactions between ATSWI3D and the Arabidopsis homologs of SWI2/SNF2 ATPases.
ATSWI3A and ATSWI3B Are Essential for
Embryonic Development
Our protein interaction studies suggested that ATSWI3A or
ATSWI3B, together with BSH/SNF5, could represent a selective
core of CRCs, which may recruit ATSWI3C or ATSWI3D as well
as different SNF2-like ATPases to regulate specific chromatinassociated functions. This model suggests two different testable
predictions. The first is that ATSWI3A and ATSWI3B are functionally equivalent and therefore redundant. Hence, in the
absence of ATSWI3A, the equivalent ATSWI3B CRC subunit
could recruit all other SWI/SNF components to perform the same
functions. By contrast, the second prediction is that ATSWI3A
and ATSWI3B perform specific and possibly essential functions.
Thus, the functions of ATSWI3 subunit combinations could be
specific to different regulatory pathways. In this study, we found
that both atswi3a and atswi3b mutations result in lethality by
arresting embryo development during the globular (blastocyte)
stage. This observation indicated that the ATSWI3A and ATSWI3B genes perform essential and nonredundant functions
required for early development. However, whether similar
phenotypes of atswi3a and atswi3b mutations imply that a SWI3
complex (AB or AA or BB) is required only for embryogenesis is
a question that should be answered by examining the effects of
these mutations in somatic cells (i.e., using genetic mosaics).
Inhibition of ATSWI3B expression by an antisense approach was
reported to cause leaf curling, dwarfism, and delayed flowering,
suggesting that ATSWI3B also performs important regulatory
functions in sporophytic tissues (Zhou et al., 2003). However, it is
unclear whether this antisense approach was specific for ATSWI3B or also influenced the expression of other ATSWI3 homologs. The latter possibility is suggested by phenotypic similarities
between ATSWI3B-silenced plants and the atswi3c and atswi3d
insertion mutants.
The phenotypes of Arabidopsis atswi3a and atswi3b mutations
are comparable to those of knockouts in the mouse Srg3 and
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The Plant Cell
Caenorhabditis psa1 SWI3 homologs. These and other mutations, affecting the mouse SWI/SNF subunits Brg1 and SNF5/
Ini1, all result in early embryonic death. Heterozygous Brg1þ/
and SNF5/Ini1þ/ animals show predisposition to tumor formation, indicating that SWI/SNF complexes found in direct association with Rb (the retinoblastoma tumor suppressor) play a key
role in controlling cell cycle progression (Kim et al., 2001; Cui
et al., 2004; Roberts and Orkin, 2004). Null mutations inactivating
the Arabidopsis Rb-related RBR1 gene result in embryonic
defects before or during the globular stage. The rbr1 mutation
does not prevent normal megagametogenesis and allows the
formation of a normal embryo sac. However, in emasculated
rbr1/þ flowers, ;25% of the ovules show fertilization-independent
cell division, producing supernumerary nuclei at the micropylar
pole. The rbr1 allele is not transmitted maternally and cannot be
rescued by paternal RBR1, as a result of a general absence of
expression of some paternal genes in the embryos until the late
globular stage. The rbr mutant alleles also show greatly reduced
paternal transmission and result in the formation of degenerated
pollen (Ebel et al., 2004).
In contrast with the rbr1 mutation, the atswi3a and atswi3b
mutations do not prevent female transmission and can be
rescued in the female by the paternal wild-type allele. The effects
of these mutations on embryo development differ slightly,
because the atswi3b mutation results in cell divisions with
aberrant polarity in the suspensor, which is not seen in the
atswi3a mutants. A remarkable effect of the atswi3b mutation is
that after pollination of atswi3b/þ plants with the wild type,
;50% of the progeny are represented by arrested ovules, and
the viable offspring show a 1:1 transmission of the atswi3b allele.
We found that the arrested-ovule phenotype is caused by the
absence of embryo sac formation and the lack of mitotic
divisions of the megaspore. This arrested-ovule phenotype is
observed in two different insertion mutants, segregates independently of the atswi3b alleles, and shows a non-Mendelian
inheritance, suggesting atswi3b-dependent imprinting of a yet
unknown locus. An experimental difficulty was revealed by the
reciprocal cross, in which we pollinated wild-type females with
atswi3b/þ males. Namely, these crosses consistently produced
unfertilized ovules, the phenotype of which could not be easily
distinguished by visual screening from that of arrested ovules
resulting from imprinting. Therefore, we examined the effect of
the atswi3b mutation on the process of microsporogenesis and
found that ;50% of microspores underwent degeneration after
the formation and separation of tetrads. Yet, the wild-type
progeny from the reciprocal crosses segregated the atswi3a
and wild-type alleles at a 1:1 ratio. Thus, degeneration of ;50%
of microspores in males and 50% of macrospores in females
could only be attributed to a novel imprinting effect caused by the
atswi3b mutation. As no similar imprinting effect was observed in
the atswi3a mutants, this trait appeared to be specific for the
atswi3b mutant alleles.
Novel Aspects in the Organization of Plant
SWI/SNF-Type Complexes
The data discussed above demonstrate that the SWI3 proteins
are of similar importance for critical developmental functions in
both animals and plants. However, there may be considerable
differences between the two kingdoms in the details of the
mechanisms in which these proteins are involved. The plant
SWI3-type proteins have clearly evolved into forms that are
functionally much more diversified than their animal counterparts. All SWI/SNF complexes characterized to date in other
eukaryotes carry bromodomain-type SWI2/SNF2 ATPases as
central catalytic subunits. We have shown that members of the
Arabidopsis SWI3 A/B subfamily are required for embryo viability, similar to their mammalian homologs. Yet, our preliminary
data indicate that a null mutation of the only Arabidopsis
bromodomain-type SWI2/SNF2 ATPase, BRAHMA, does not
cause embryo lethality (K. Brzeska and M. Prymakowska-Bosak,
personal communication). This would indicate that BRAHMA
may not be part of SWI/SNF complexes that carry only the
essential ATSWI3A or ATSWI3B subunits. Rather, BRAHMA may
be part of an ATSWI3C-based SWI/SNF complex, as suggested
by phenotypic similarities between atswi3c mutants and
BRAHMA-silenced plants. Because the bromodomain is thought
to increase the processivity of SWI/SNF-mediated nucleosome
remodeling through its affinity for acetylated histones, the lack of
a bromodomain could greatly affect the interactions between
ATP-dependent remodeling complexes and epigenetic chromatin marks. Why would a canonical SWI/SNF complex carrying the
only known Arabidopsis bromodomain ATPase be excluded
from the control of essential functions in plants? Is the acetylated histone binding function of plant SWI2/SNF2 ATPases taken
over by other unknown domains or associated with noncore
subunits? The answers to these questions require further identification and characterization of the components of different
SWI3-containing CRCs in plants.
METHODS
Plant Material and Growth Conditions
The atswi3a-1 (SALK_035320), atswi3a-2 (SALK_065548), atswi3a-3
(SALK_068234), and atswi3d-1 (SALK_100310) mutant alleles were
identified in Arabidopsis thaliana seed populations received from the
SALK collection (Alonso et al., 2003). Using a PCR-based screening
strategy, the atswi3b-1 (Koncz_2208), atswi3c-1 (Koncz_27320), atswi3c-2
(Koncz_3737), and atswi3d-2 (Koncz_14259) mutant alleles were identified in our T-DNA mutant collection (Rı́os et al., 2002). The atswi3b-2
(GABI_302G08) mutant allele was obtained from the GABI-Kat T-DNA
mutant population (Rosso et al., 2003). For germination, seeds were
surface-sterilized with 5% calcium hypochlorite containing 0.01% (v/v)
Tween 20, rinsed three times with sterile water, and plated on Murashige
and Skoog (MS) seed germination medium containing 0.5% sucrose as
described (Koncz et al., 1994). Segregation of antibiotic resistance
markers encoded by the T-DNA tags was assayed by growing seedlings
in MS medium containing 15 mg/L hygromycin (our lines), 50 mg/L kanamycin (SALK lines), or 12 mg/L sulfadiazine (4-amino-N-[2-pyrimidinyl]
benzene-sulfonamide-Na; GABI-Kat lines).
Segregation analysis of the SALK lines was performed with a limited
number (30 to 40) of seedlings by PCR as described by Rı́os et al. (2002).
From each identified heterozygous atswi3a line, the genotypes of 30
offspring obtained after self-pollination were determined by PCR. In
addition, 20 to 30 siliques were opened on each atswi3a/þ plant to
determine the segregation ratio of wild-type seeds (wt) and aborted seeds
Control of Development by SWI3 Proteins
(as). The observed segregation ratios were as follows: atswi3a-1/þ,
2.95wt:1as (x2 ¼ 0.0896, P ¼ 0.8); atswi3a-2, 3.02wt:1as (x2 ¼ 3.85 3
103, P ¼ 0.95); and atswi3a-3, 3.008wt:1as (x2 ¼ 1.85 3 104, P ¼ 0.97).
The segregation ratios of antibiotic-resistant (R) versus antibiotic-sensitive (S) progeny of atswi3b mutants were as follows: atswi3b-1, 2.1R:1S
(x2 ¼ 0.55, P ¼ 0.5); and atswi3b-2, 1.77R:1S (x2 ¼ 1.689, P ¼ 0.22). The
original GABI_302G08 line carrying the atswi3b-2 allele contained two
independently segregating T-DNA insertions, which were separated by
repeated outcrosses with the wild type. The segregation of wild-type,
aborted-seed, and arrested-ovule traits of self-pollinated atswi3b/þ
mutants is shown in Table 1. The segregation ratios of hygromycinresistant to hygromycin-sensitive progeny of the atswi3c and atswi3d
alleles were as follows: atswi3c-1, 2.645R:1S (x2 ¼ 2.01, P ¼ 0.16);
atswi3c-2, 3.307R:1S (x2 ¼ 0.0952, P ¼ 0.76); and atswi3d-2, 2.75R:1S
(x2 ¼ 4.2 3 102, P ¼ 0.84). PCR screening for the atswi3d-1 allele
indicated a segregation of 0.9:2.07:0.98 (wt:atswi3c-2/þ:atswi3d-2/
atswi3d-2; x2 ¼ 0.163, P ¼ 0.9). The x2 and P values indicate deviations
from the expected 3:1, 2:1, or 1:2:1 segregation ratios and suggest a potential reduction of male or female transmission.
Analysis of T-DNA Insert Junctions in the Insertion Mutant Lines
We screened for T-DNA insertions in all ATSWI3 genes using PCR
with pairs of gene-specific and T-DNA end–specific primers. The genespecific primers were used in subsequent PCRs to classify the
heterozygous and homozygous mutant lines. After isolation of the
PCR-amplified T-DNA–plant DNA junction fragments from the screening
gels, their sequences were determined. In the sequences provided for the
T-DNA junctions of each mutant allele below, uppercase letters mark
plant sequences and lowercase letters mark T-DNA sequences. The
atswi3a-1 allele was identified using combinations of gene-specific
FSSB1 (59-CCGTGTGGTTTGGATTTGGCGATTG-39) and FSSB2
(59-CGCGTGTGAATTAGTAGAGAGACCCA-39) primers with the T-DNA
end primers LBb1 (59-GCGTGGACCGCTTGCTGCAACT-39) and TDNAUSALK (59-GATGAGACCTGCTGCGTAAG-39). In atswi3a-1, the left border
junction of the T-DNA (LB in Figure 3) was 59-GGCAGGGTATAttgtggtgtaaacaaa-39. The right border junction carried a deletion of 125 bp in the
T-DNA, and the junction with the truncated T-DNA end was 59-tttaaactatcagtgtttaaacACTATGGGGAGAACAA-39. The T-DNA insertions in
atswi3a-2 and atswi3a-3 mutants were confirmed by PCR using the
gene-specific primers FSSB1 and FSSB2 and the T-DNA–specific primer
LBb1. In the atswi3a-2 mutant allele, the left borders (LB1 and LB2) of an
inverted T-DNA repeat were linked to plant DNA (Figure 3). The sequence
of the LB1 junction was 59-CTCCTGCTGGAAcaaattgacgctt-39, whereas
that of the LB2 junction was 59-gcgtcaacctcgtTTGAGGAAAGAG-39. The
atswi3a-3 mutant allele similarly carried an insert of inverted T-DNA
repeat, the ends of which were designated LB3 and LB4. The sequence
corresponding to LB3 was 59-GCGCTAGAATCTGTGacacaaattgacgctt39 and that of LB4 was 59-ccatgtgtaatttgttTACCAAGAGTAG-39.
T-DNA tags in the ATSWI3B gene were identified as described above
using the gene-specific primers FSSA1 (59-CTTCTCCGGCGAAGTTGCGTTAGTT-39) and FSSA2 (59-CTCCAATTGTTTCCGGCTTCTCTCCAT39) together with the T-DNA–specific primer FISH1 (59-CTGGGAATGGCGAAATCAAGGCATC-39) for our mutant collection. The T-DNA insertion
in the atswi3b-2 allele was identified using the gene-specific primers
FSSA1 and FSSA2 together with the T-DNA–specific primer for the GABIKat collection, GABI-LEFT (59-TCTCCATATTGACCATCATACTCATTGC-39). The junctions of an inverted T-DNA repeat in the atswi3b-1
mutant allele (Figure 3) were LB1 (59-AGTCAAAGCCAGgatatattcaat-39)
and LB2 (59-ctcattgctcatTGTTATGCACTA-39). The atswi3b-2 mutant
allele carried a similar inverted T-DNA repeat, the junctions of which
were LB3 (59-GAGAGTCATGGCtaccgaaaaattgataaaatga-39) and LB4
(59-aatatatcctgaCACTCCCTCTCT-39). The positions of the ATG codon
2469
of AtSWI3B and an in-frame stop codon in the T-DNA sequence are
underlined.
To identify T-DNA tags in the ATSWI3C gene, we used the genespecific primers FSSC1 (59-AATGCGCGACGGTCGACTAATGTATC-39)
and FSSC2 (59-AGCCTGAACCTGTGGAAGACCTAAC-39) in combination
with the T-DNA left border primer FISH1 and the RB primer FISH2
(59-CAGTCATAGCCGAATAGCCTCTCCA-39). The junction sequences
of the atswi3c-1 allele (Figure 3) were as follows: right border junction (59-AGATTCTCCTCTtattttatttttatt-39) and left border junction (59-tccttatgtaggataataaTGGTGCCACA-39). The junction sequences of the inverted T-DNA tag in the atswi3c-2 mutant allele were LB1 (59-ATAAATTCTCTGTAGTatatattcaattg-39) and LB2 (59-aattgaatatatcCTGAAACTTCTT-39).
To identify the T-DNA insertion in the ATSWI3D gene, PCR screening
was performed with combinations of the gene-specific primers WTDU
(59-GGCGTTGGTAGTGGGAAGTGGA-39) and WTDL (59-TCTGGTTCTGGAACTTCTTTCA-39) with the T-DNA end primer LBb1 for the SALK
collection. To screen our insertion mutant collection, we used the genespecific primers AtSWI3D1 (59-AGAAGCGAAATTGGGGGATTGGGAATAG-39) and AtSWI3D2 (59-AGGTTGAAGCCGCAAAAGGTGGGATAAT-39) in combination with the T-DNA end primer HOOK1 (59-CTACACTGAATTGGTAGCTCAAACTGTC-39). The insertions in both atswi3d-1
and atswi3d-2 alleles represented inverted T-DNA repeats facing the
plant DNA with their left borders. The junction sequences of the T-DNA insert in atswi3d-1 were LB1 (59-CACCTGAAGAGGacaacttaataaca-39) and
LB2 (59-aagcgtcaatttgttattAAGATGAAACTATG-39), whereas in atswi3d-2
they were LB3 (59-CAGAAGAGGTCaatatggatcagc-39) and LB4 (59-gacatgaagccatTTCAGATGATAG-39).
Assays of Flowering Time during SD and LD Conditions
Segregating families of atswi3c-1/þ, atswi3c-2/þ, atswi3d-1/þ, and
atswi3d-2/þ mutant lines were planted into soil and grown either under
SD conditions using an 8-h light/16-h dark cycle or under LD conditions
with a 16-h light/8-h dark cycle. During the day, temperature was
a constant 238C with 60% humidity, whereas the night temperature was
208C with 70% humidity. The intensity of irradiance was 200 mEm2s1.
Samples for RNA isolation were collected under LD conditions at midday
(8 h after dawn) from seedlings at day 15 after sowing and from
inflorescence tips on the day of appearance of the first visible flower
bud. Under SD conditions, samples were taken 6 h after dawn at days 15,
31, and 41 from seedlings and on the first day of flowering (i.e., day 66,
which overlapped in the flowering time schedules of the atswi3c and
atswi3d mutants and the wild type).
Analysis of mRNA Levels in the atswi3c and atswi3d Mutants
Total RNA was isolated from 100 mg of tissue of atswi3c, atswi3d, and
wild-type plants using the RNeasy plant mini kit (Qiagen). One microgram
of total RNA was reverse-transcribed using the Transcriptor First Strand
cDNA synthesis kit (Roche) according to the manufacturer’s instructions.
Equal aliquots from each RT reaction were used as template to amplify
the ATSWI3C and ATSWI3D cDNAs shown in Figures 7A and 9A.
Amplification of full-length ATSWI3C cDNA was performed with the
gene-specific primers SWI3C1 (59-TCCCCCGGGGCCAGCTTCTGAAGAT-39) and SWI3C2 (59-ACGCGTCGACTAGTTTAAGCCTAAGCCGGA-39) using denaturation at 958C for 5 min, followed by 35 cycles of
958C for 30 s, 568C for 30 s, and 728C for 2 min, and elongation at 728C for
10 min. Amplification of full-length ATSWI3D cDNA was performed with
the primers SWI3D1 (59-ATGGAGGAAAAACGACGCGATT-39) and
SWI3D2 (59-CTAAACCGAAGAACATTGTCTG-39) using denaturation at
958C for 5 min, followed by 35 cycles of 958C for 30 s, 568C for 30 s, and
728C for 4 min, and extension at 728C for 10 min.
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The Plant Cell
For RT-PCR analysis of flowering time integrator and floral homeotic
genes, cDNA templates were prepared from two independently isolated
sets of mRNAs. PCR was performed after denaturation at 958C for 5 min
using 22, 30, or 35 cycles of amplification (958C for 30 s, 568C for 30 s, and
688C for 1 min) followed by an elongation step at 688C for 5 min. The
following pairs of gene-specific primers were used: AP1-1 (59-acaagtgacattctcgaaaagaaga-39) and AP1-2 (59-tggactcgtacataagttggtttttc-39); AP2-1
(59-cgattatgatgatgacttgaaacag-39) and AP2-2 (59-cttgacttttatgcttcgaattagc-39); AP3-1 (59-atgagtatatcagccctaacaccac-39) and AP3-2 (59-gagtcgtaatctcctccattgtcta-39); PI-1 (59-gatgattgattactgttgtccttcc-39) and PI-2
(59-atcatgatctctcatcatcattcct-39); AG-1 (59-agaaagcttacgagctctctgttct-39)
and AG-2 (59-cgttatgcaaatcaacttctctttt-39); FLC-1 (59-atcaagcgaattgagaacaaaagta-39) and FLC-2 (59-attctcaacaagcttcaacatgagt-39); FT-1 (59-atagtaagcagagttgttggagacg-39) and FT-2 (59-tgtcgaaacaatataaacacgacac-39);
CO-1 (59-tgaatacagtcaacaccaacaaaac-39) and CO-2 (59-tgtcttctcaaatttccttgtcttc-39); SOC1-1 (59-agtgactttctccaaaagaaggaat-39) and SOC1-2
(59-ctgctcaatttgttccttaaacact-39); UFO-1 (59-tctctcttttgcttatatcccttca-39)
and UFO-2 (59-cgctaaaagggctatagttcataca-39); LFY-1 (59-gtccgtacggtatacgtttctacac-39) and LFY-2 (59-gttgcttcttcatctttccttgac-39); and Actin2
control ACT1 (59-agagattcagatgcccagaagtcttgtcc-39) and ACT2 (59-aacgattcctggacctgcctcatcatactc-39).
Cytological Techniques
Immature seeds were removed from green siliques at various times after
pollination with a dissecting microscope and cleared in a small amount of
Hoyer’s solution (30 mL of water, 100 g of chloral hydrate, 7.5 g of gum
arabic, and 5 mL of glycerin) on a glass slide. Mutant seeds usually
cleared in 4 to 12 h, depending on their developmental stage, and were
examined with a compound microscope equipped with Nomarski optics.
To prepare cross sections of leaves and flowers, regular segments of 2 to
3 mm were cut from the same position of leaves and flower buds. The
sections were fixed for 2 h in 2% glutaraldehyde in 0.1 M cacodylate
buffer, pH 7.2, dehydrated using a graded series of ethanol and propylene
oxide, and embedded in Spurr resin (Spurr, 1969). Alternatively, the
sections were fixed in 4% glutaraldehyde in 50 mM PIPES buffer, pH 7.1,
washed with the buffer three times for 15 min, and postfixed in 1%
osmium tetroxide for 24 h in the same buffer. After washing and dehydration in acetone, the samples were infiltrated and embedded in Spurr
medium. Semithin sections of 1 to 2 mm were cut using a Leica RM2065
microtome or an LKB ultramicrotome, stained with 0.1% toluidine blue in
1% borax or by basic fuchsine in combination with toluidine blue or
methylene blue, as described by Farrás et al. (2001), and observed with
a Leica Aristoplan light microscope using a Hitachi HV-20 camera for
image recording. For scanning electron microscopy, the samples were
processed with an electron microscope cryopreparation system
(CT1500; Oxford Instruments) and examined with a Zeiss DSM 940
microscope.
Protein Interaction Assays in the Yeast Two-Hybrid System
Plasmids pGADSWI3A, pGADSWI3C, pGBSH, pGADFCA, and pFCATRUNCATED used in yeast two-hybrid assays were described previously
(Sarnowski et al., 2002). Plasmids pGBTSWI3A and pGBTSWI3C were
obtained by cloning full-length ATSWI3A and ATSWI3C cDNAs from
pGADSWI3A and pGADSWI3C, respectively, in pGBT9 (DNA-BD) using
SmaI and BamHI. A full-length ATSWI3D cDNA was amplified using PCR
with primers SWI3D1 and SWI3D2 (see above) and cloned into the TOPO
2.1 cloning vector (Invitrogen). Upon sequencing, the ATSWI3D cDNA
was moved by EcoRI into the vectors pGBT9 and pGAD424, yielding
pGBTSWI3D and pGADSWI3D, respectively. Plasmid pCL1 was used as
a positive control (Sarnowski et al., 2002). Yeast strain Y190 was
transformed with the following plasmid pairs: pGBSH-pGADSWI3A,
pGBSH-pGADSWI3C, pGBTSWI3A-pGADSWI3A, pGBTSWI3A-pGADSW-
I3C, pGBTSWI3A-pGADFCA and pGBTSWI3A-pFCATRUNCATED (FCAPhD-AD), pGBTSWI3A-pGADSWI3D, pGBTSWI3B-pGADSWI3D, pGBSHpGADSWI3D, as well as with the control plasmids pGBSH, pGBTSWI3A,
pGADSWI3A, pGBTSWI3C, pGADSWI3C, pFCATRUNCATED, pGADFCA,
pGBTSWI3D, pGADSWI3D, and pCL1 in combination with either pGBT9 or
pGAD424. All transformants were grown in selection medium containing 100
mM 3-amino-1,2,4-triazole, and the level of b-galactosidase activity was
monitored by the replica filter lift method described in the Clontech yeast
protocol handbook.
Sequence Homology Searches and Phylogenetic Tree Analysis
Arabidopsis and rice (Oryza sativa) SWI3 homologs were identified using
the National Center for Biotechnology Information tBLASTN search
facility and the Oryza database (http://riceblast.dna.affrc.go.jp) and
were compared with entries in the Plant Chromatin Database (http://
chromdb.org). Multiple alignments were generated using the ClustalW
program package at the Pasteur Institute (http://bioweb.pasteur.fr/),
including all correction and statistical control facilities for improving the
sensitivity, weighting, and position-specific gap penalties. The multiple
alignments were used for phylogenetic analyses. The tree consensus was
generated either by the quartet puzzling maximum likelihood method
(Strimmer and von Haeseler, 1996) or by generating a consensus maximum parsimony tree using the PHYLIP package (Felsenstein, 1989). The
bootstrap analysis was based on 1000 replications.
Accession Numbers
Sequence data from this article can be found in the GenBank/EMBL data
libraries under accession numbers AY081570 (ATSWI3A), NM_128921
(ATSWI3B), AY091026 (ATSWI3C), NM_202953 (ATSWI3D), NM_112639
(BSH), and Z82989 (FCA).
ACKNOWLEDGMENTS
We thank Sabine Schäfer, Andrea Lossow, Christine Gerdes, and Ingrid
Reintsch for their help in the insertion mutant screens. The Polish
laboratory was supported by the Center of Excellence for Multiscale
Biomolecular Modeling, Bioinformatics, and Applications. T.J.S. was
supported by a Federation of European Biochemical Societies ShortTerm Fellowship (2004). S.S. was supported by the British Council and
Polish Ministerstwo Nauki i Informatyzacji Young Scientists Programme.
This work was supported by Howard Hughes Medical Institute Grant
55000312 and Polish Committee for Scientific Research Grant PBZ-039/
PO4/2001 to A.J., by European Union Grants QLK-5-200101871 and
QLK5-CT-2002-00841 and Deutsche Forschungsgemeinshaft Grant
AFGN Ko 1438/9-1 to C.K., and by a Marie Curie Fellowship (EU
HPMF-CT-2000-00597) to G.R.
Received January 28, 2005; revised June 15, 2005; accepted June 15,
2005; published July 29, 2005.
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