Download FIE and CURLY LEAF polycomb proteins interact

The Plant Journal (2004) 37, 707±719
doi: 10.1111/j.1365-313X.2003.01996.x
FIE and CURLY LEAF polycomb proteins interact in the
regulation of homeobox gene expression during sporophyte
development
Aviva Katz, Moran Oliva, Assaf Mosquna, O®r Hakim and Nir Ohad
Department of Plant Sciences, Tel Aviv University, Tel Aviv 69978, Israel
Received 22 September 2003; revised 20 November 2003; accepted 26 November 2003.
For correspondence (fax ‡972 3 640 9380; e-mail [email protected]).
Summary
The Arabidopsis FERTILIZATION-INDEPENDENT ENDOSPERM (FIE) polycomb group (PcG) protein, a WD40
homologue of Drosophila extra sex comb (ESC), regulates endosperm and embryo development and
represses ¯owering during embryo and seedling development. As ®e alleles are not transmitted maternally,
homozygous mutant plants cannot be obtained. To study FIE function during the entire plant life cycle, we
used Arabidopsis FIE co-suppressed plants. Low FIE level in these plants produced dramatic morphological
aberrations, including loss of apical dominance, curled leaves, early ¯owering and homeotic conversion of
leaves, ¯ower organs and ovules into carpel-like structures. These morphological aberrations are similar to
those exhibited by plants overexpressing AGAMOUS (AG) or CURLY LEAF (clf ) mutants. Furthermore, the
aberrant leaf morphology of FIE-silenced and clf plants correlates with de-repression of the class I
KNOTTED-like homeobox (KNOX ) genes including KNOTTED-like from Arabidopsis thaliana 2 (KNAT2 )
and SHOOTMERISTEMLESS (STM ), whereas BREVIPEDICELLUS (BP ) was upregulated in FIE-silenced
plants, but not in the clf mutant. Thus, FIE is essential for the control of shoot and leaf development. Yeast
two-hybrid and pull-down assays demonstrate that FIE interacts with CLF. Collectively, the morphological
characteristics, together with the molecular and biochemical data presented in this work, strongly suggest
that in plants, as in mammals and insects, PcG proteins control expression of homeobox genes. Our
®ndings demonstrate that the versatility of the plant FIE function, which is derived from association with
different SET (SU (VAR)3-9, E (Z), Trithorax) domain PcG proteins, results in differential regulation of gene
expression throughout the plant life cycle.
Keywords: CLF, FIE, homeobox genes, KNOX genes, polycomb proteins, sporophyte development.
Introduction
The alternation between the sporophyte and gametophyte
phases of the life cycle of higher plants requires the activation and repression of appropriate developmental programmes. FERTILIZATION-INDEPENDENT ENDOSPERM
(FIE) protein is an important regulator of reproductive
programmes in plants. Mutations in the Arabidopsis FIE
gene affect the female gametophyte, allowing the central
cell to replicate and differentiate into endosperm, in the
absence of fertilization, without triggering the development
of the egg cell into an embryo (Ohad et al., 1996). FIE
encodes a WD40-type protein, homologous to the Drosophila polycomb group (PcG) protein, extra sex comb (ESC)
(Ohad et al., 1999). In plants, as in insects and mammals,
PcGs are involved in the regulation of various developmenß 2004 Blackwell Publishing Ltd
tal programmes (reviewed by Berger and Gaudin, 2003;
Chaudhury et al., 1998; Goodrich and Tweedie, 2002; Hsieh
et al., 2003; Kohler and Grossniklaus, 2002; Sung et al.,
2003; Wagner, 2003). In insects and mammals, the WD40type protein ESC and its orthologue embryonic ectoderm
development (EED) form complexes containing SET
domain protein (Jones et al., 1998; Sewalt et al., 1998;
Tie et al., 1998). These complexes bind to and alter chromatin condensation, resulting in downregulation of
homeotic target gene expression (reviewed by Simon
and Tamkun, 2002). Similarly, interactions between the
Arabidopsis SET domain PcG protein MEDEA (MEA) and
FIE have been shown to take place in vitro (Luo et al., 1999;
Spillane et al., 2000; Yadegari et al., 2000), indicating that
707
708 Aviva Katz et al.
FIE and MEA form a PcG complex in vivo, regulating endosperm and embryo development as was recently shown
(Kohler et al., 2003a). FIE was found to repress the expression of MADS-box gene family members (MINICHROMOSOME MAINTENANCE 1 (MCM1) genes in yeast,
AGAMOUS (AG ) in Arabidopsis, DEFICIENS (DEF ) in Antirrhinum and serum response factor (SRF ) in humans)
(Riechmann and Meyerowitz, 1977), and to prevent seedlings from ¯owering precociously (Kinoshita et al., 2001).
Similarly, EMBRYONIC FLOWER2 (EMF2) and VERNALIZATION2 (VRN2), homologues of Su(z)12, a subunit of the
Drosophila ESC protein complex, control ¯owering through
the regulation of MADS-box genes (Gendall et al., 2001;
Moon et al., 2003). FIE, MEA and FERTILIZATION-INDEPENDENT SEED2 (FIS2) were shown to control expression of
PHERES1 (PHE1), a type I MADS-box gene, which regulates
seed development. Furthermore, FIE and MEA associate
with the promoter region of PHE1 (Kohler et al., 2003b).
Thus, whereas PcG proteins in plants control MADS-box
genes, in mammals and insects they control homeotic
genes, which belong to the homeobox gene family.
The expression of FIE mRNA in all wild-type Arabidopsis
tissue during the vegetative and reproductive phases (Ohad
et al., 1999) implies a possible role for FIE throughout the
entire plant life cycle. The question arises as to what programmes are controlled by the FIE±PcG complex in the
sporophyte, and what proteins take part in such complex.
Until recently, it was not possible to study FIE function in
the sporophyte, as mutant ®e alleles are not transmitted
through the female gamete, thus preventing the formation
of homozygous mutant plants (Ohad et al., 1996). Using a
pFIE:FIE-GFP (GREEN FLUORESCENT PROTEIN) transgene,
it was possible to partly rescue ®e mutant embryos, which
were able to germinate, but failed to develop into an adult
plant. Occasionally, however, seedling shoots, hypocotyls
and roots produced ¯ower-like structures and organs,
demonstrating that FIE is required to suppress the ¯oral
programme in the early stages of plant development
(Kinoshita et al., 2001).
To explore the role of FIE during sporophyte development and understand how it functions, we characterized
Arabidopsis transgenic plants co-suppressed for FIE
expression. Signi®cantly reduced levels of FIE protein
caused pleiotropic aberrant phenotypes, while still allowing the plants to reach maturity. Analysis of the plants
revealed that FIE controls leaf development by regulating
members of the homeobox and the MADS-box gene
families. We also found that FIE±PcG protein interacts with
CURLY LEAF (CLF), a SET domain PcG protein known to
regulate leaf and ¯ower differentiation (Goodrich et al.,
1997). The above results imply that FIE may associate with
different SET-domain proteins to form PcG complexes
controlling gametophytic and sporophytic developmental
programmes.
Results
Ectopic expression of GFP:FIE resulted in FIE
co-suppression
Transgenic plants were generated to express a translational
fusion between GFP and FIE cDNA, driven by the cauli¯ower mosaic virus (CaMV) 35S promoter. Seven out of 60
independent transgenic T1 plants displayed similar abnormal phenotypes, including loss of apical dominance,
abnormal rosette leaves, curled cauline leaves, early ¯owering, homeotic conversions of ¯ower organs and delayed
senescence. Southern blot analysis of genomic DNA,
extracted from T3 plants of the above seven lines, using
FIE cDNA as a probe, revealed insertions into several different loci (data not shown), indicating that the observed
abnormal phenotypes did not result from the disruption of
a speci®c locus. T3 homozygous transgenic plants segregated to plants displaying either abnormal or normal
phenotypes. Expression of the GFP-FIE transgene and
endogenous FIE in the above T3 segregating plants was
tested using semiquantitative RT-PCR. The expression
levels of the GFP-FIE transgene were signi®cantly lower
in the abnormal transgenic plants as compared to transgenic plants exhibiting the normal phenotype (Figure 1a).
The expression level of the endogenous FIE was similar in
wild-type plants and transgenic segregates with normal
phenotypes. However, transcript level of the endogenous
FIE was signi®cantly lower in the abnormal transgenic
segregates (Figure 1a). The reduction in endogenous and
transgenic FIE expression levels was observed in both
vegetative (rosette leaves) and reproductive tissue (cauline
leaves and in¯orescences). The correlation between abnormal phenotypes and reduced expression of both endogenous and transgenic FIE indicated that the abnormal
phenotype was caused by co-suppression of FIE expression
(Denli and Hannon, 2003; Szweykowska-Kulinska et al.,
2003). Furthermore, among some of the transgenic lines,
few plants displayed chimaeric phenotypes producing normal and abnormal branches. This further supports the
conclusion that silencing resulted from transgene-induced
co-suppression. Western analyses performed with af®nitypuri®ed antibodies against FIE have shown that FIE is not
detected in the rosette leaves of abnormal transgenic
plants, but does appear in wild-type leaves, con®rming
the above conclusion (Figure 1b). Thus, we refer to the
abnormal GFP:FIE transgenic plants as `FIE-silenced plants'.
FIE-silenced plants display pleiotropic phenotypes
The FIE-silenced plants exhibited a range of abnormal
phenotypes as shown in Figure 2. Loss of apical dominance
was evident from the formation of six to eight stems
as compared to one to three stems in wild-type plants
ß Blackwell Publishing Ltd, The Plant Journal, (2004), 37, 707±719
FIE regulates homeobox gene expression in sporophyte development
Figure 1. Expression of FIE in wild-type and FIE-silenced plants.
(a) Ethidium bromide-stained gel of RT-PCR products. Total RNA extracted
from wild-type (wt) and GFP:FIE transgenic plants displaying either normal
(normal) or abnormal phenotypes (abnormal) served as template. RT-PCR
was performed on RNA samples extracted from rosette, cauline leaves and
in¯orescences using primers that identi®ed either the endogenous FIE or the
GFP:FIE transcripts. Ampli®cation of ubiquitin (UBI) was used as internal
control.
(b) Detection of FIE by Western analysis of protein extracts from wt and FIEsilenced rosette leaves using anti-FIE antibodies (left panel). Equivalent
amounts of protein extracts were loaded as determined by staining the
membrane with Ponceau-red (right panel).
(Figure 2a,b). Occasionally, stems were fasciated (Figure 2e,f),
indicating enlargement of the shoot apical meristem.
Whereas FIE-silenced and wild-type plants developed the
same number of internodes along the in¯orescence stem,
in FIE-silenced plants the internodes were shorter, resulting
in stunted plants (Figure 2b). Under long photoperiod (16 h
light/8 h dark), FIE-silenced plants ¯owered, on average,
21 days after germination, exhibiting eight rosette leaves,
while wild-type plants ¯owered, on average, 27 days after
germination, producing 11±13 rosette leaves. These results
support earlier ®ndings indicating that FIE suppresses ¯owering during the late stages of embryogenesis (Kinoshita
et al., 2001), revealing a similar function of FIE during the
vegetative phase of development. The ®rst four to six
rosette leaves of FIE-silenced plants (part of the vegetative
phase) exhibited normal morphology, while the successive
rosette leaves were narrower than the wild type, rolled up
towards the midrib and displayed varying degrees of serration (Figure 2c,h). Occasionally, stipules were longer than
those of wild-type plants, and developed stigmatic-like
ß Blackwell Publishing Ltd, The Plant Journal, (2004), 37, 707±719
709
papillae at the tip (Figure 2g). Cauline leaves were smaller
than those of wild-type plants, and their margins rolled up
towards the midrib (Figure 2i,j). Occasionally, stigmaticlike papillae and initials of ovule-like primordia developed
at the marginal tips of cauline leaves (Figure 2j), indicating
a homeotic conversion to carpel-like organs. The in¯orescences produced fewer ¯owers than those in wild-type
plants, and often developed terminal ¯owers in which the
outer whorls (sepals and petals) formed carpeloid-like
organs (Figure 2o). Stem fasciations caused ¯ower clustering and loss of normal phylotaxy (Figure 2e,f). Flower
organ abnormalities were observed in all four whorls
(Figure 2k±s). The ®rst whorl had narrow sepals, white at
their tips and occasionally serrated (Figure 2p). The second
whorl had narrower petals (Figure 2q), often emerging
between unopened sepals (data not shown). Frequently,
stamenoid petals (Figure 2q) and petaloid stamens
(Figure 2r) developed, resulting in variable numbers of
stamens, ranging from ®ve to seven. In addition, stamens
often produced a reduced amount of pollen. Some stamens
developed stigmatic-like papillae at their tips (Figure 2o). In
most ¯owers of the FIE-silenced plants, the ovaries were
misshapen, comprising three fused carpels (Figure 2s).
Occasionally, several carpels were fused to form a complex
¯ower (Figure 2f). FIE-silenced ovules often had a longer
funiculus, and some developed stigmatic papillae at their
tips and subtending ovules at their margins, indicating a
homeotic transformation of the ovules into a carpel-like
structure (Figure 3). The fertility of FIE-silenced ¯owers was
low, and ¯oral organs tend not to detach after anthesis
(Figure 2b). Plant senescence was also found delayed, possibly because of a reduction in fertilization, as observed in
various male sterile mutants (Chaudhury et al., 1994). Thus,
the reduction in FIE levels (Figure 1) interfered with a wide
range of developmental processes, demonstrating the central role of FIE as an important regulator during plant
vegetative and reproductive development.
KNOX genes are de-repressed in FIE-silenced plants
In Drosophila, mutations in the ESC gene result in homeotic
conversions and ectopic expression of transcription factors. Similarly, in FIE-silenced plants it is plausible that the
observed homeotic changes result from the ectopic expression of meristem and organ identity transcription factors.
The serrated leaf phenotype observed in FIE-silenced plants
suggested that FIE participates in the regulation of BREVIPEDICELLUS (BP), a member of class I KNOX genes (Ori
et al., 2000). Expression of all class I KNOX genes was
examined by RT-PCR, and showed that levels of BP, KNAT2
and SHOOTMERISTEMLESS (STM) (Figure 4) were signi®cantly higher in FIE-silenced plants than in wild-type plants.
No signi®cant differences in the expression of KNAT6 were
observed (Figure 4).
710 Aviva Katz et al.
Figure 2. Abnormal phenotypes displayed by FIE-silenced plants.
(a, b) Wild-type (wt) and FIE-silenced adult plants, respectively.
(c, d) Close-up of FIE-silenced and wt rosette leaves, respectively.
(e, f) Fasciated stem and multi-carpel ¯owers in FIE-silenced plants, respectively.
(g) Abnormal elongated stipules with stigmatic-like papillae structures at the tip.
(h) wt and FIE-silenced rosette leaves. Note the serration, size and curling of leaves.
(i, j) Cauline leaves of wt and FIE-silenced plants.
(k±o) wt (k) and different ¯owers of FIE-silenced plants (l±o). Note the carpeloid organ with stigmatic-like papillae in the ®rst whorl (o), the stamenoid petals (m)
and the multi-carpel gynoecia (n).
(p±s) Variable phenotypes of four different ¯ower organs: sepals, petals, stamens and carpels, respectively. Left panels show wt organs. Note the bifurcated petal
and petaloid sepal (p), the stamenoid petals (q), the petaloid stamens (r) and the multi-carpel gynoecia (s).
De-repression of BP was con®rmed by examining BP:bglucuronidase (GUS) expression patterns in the FIEsilenced background. Plants carrying BP promoter fused
to GUS reporter gene were crossed with FIE-silenced
plants, and the expression patterns of the reporter were
examined (Figure 5). In wild-type plants, the GUS expression pattern was similar to that reported by Ori et al.
(2000), i.e. it was found in the veins of cotyledons, in shoot
meristem and in the basal part of veins (Figure 5a,c). We
also detected expression in the hydathodes of rosette
leaves (Figure 5c). In the FIE-silenced background, GUS
expression extended to the vasculature of rosette leaves
(Figure 5b,d). The abnormal GUS staining could be
detected in both the ®rst four normal-looking rosette
leaves and in successive leaves with abnormal morphology, indicating that the severity of FIE-silenced leaf morphology may depend on the expression of additional
genes.
ß Blackwell Publishing Ltd, The Plant Journal, (2004), 37, 707±719
FIE regulates homeobox gene expression in sporophyte development
Figure 3. Homeotic transformation of FIE-silenced ovules into carpel-like
structures.
(a) Ovary with a protruding spaghetti-like ovule; Insert, close-up of carpellike ovule with stigmatic papillae.
(b) Nomarsky differential interference contrast image of a carpel-like ovule
with secondary ovules (see insert).
Scale bars are 0.2 mm.
ASYMMETRIC LEAVES1 (AS1) and AS2 are known to
negatively regulate class I KNOX genes, including BP and
KNAT2 (Byrne et al., 2002; Ori et al., 2000; Semiarti et al.,
2001). To discover whether FIE controls class I KNOX genes
via the regulation of AS1 and AS2, the steady-state RNA
levels of AS1 (Figure 4) and AS2 (data not shown) were
tested in rosette leaves and found to be unchanged, suggesting that AS1 and AS2 are not likely to function downstream to FIE.
FIE represses expression of homeotic genes
RT-PCR analysis of wild-type and abnormal rosette leaves
(Figure 4), cauline leaves and sepals (see Supplementary
Material, Figure S1) revealed that transcript levels of three
MADS-box genes, AGAMOUS (AG), APETALA3 (AP3) and
711
Figure 5. Expression of pBP:GUS in FIE-silenced plants.
(a) An 18-day-old wild-type seedling expressing pBP:GUS.
(b) An 18-day-old FIE-silenced seedling ¯owering prematurely.
(c) A detached rosette pBP:GUS wild-type leaf, exhibiting GUS expression in
the basal part of the veins and in the hydathodes.
(d) Detached rosette leaves from FIE-silenced plant expressing pBP:GUS.
The GUS staining is apparent in the vasculature of the fourth rosette leaf
displaying a normal phenotype and in a younger leaf, which is small, curled
and serrated, characteristic of abnormal FIE-silenced plants.
AGAMOUS LIKE17 (AGL17), as well as a PcG SET domain
protein MEA were signi®cantly elevated (Figure 4). The
steady-state RNA levels of PISTELLATA (PI) and AP1 were
not affected. No changes were detected in steady-state
transcript levels of known AG regulators such as WUSCHEL
(WUS) (Lenhard et al., 2001; Lohmann et al., 2001), AINTEGUMENTA (ANT ) (Krizek et al., 2000) and APETALA2 (AP2)
(Bowman et al., 1991; Drews et al., 1991; Kunst et al., 1989;
Modrusan et al., 1994). The expression level of SUPERMAN
(SUP), a regulator of AP3 (Bowman et al., 1992; Sakai et al.,
1995), was also not altered. Data for most of the genes that
showed no change in transcript levels are not shown.
Ectopic expression of AG in FIE-silenced plants promotes
malformation of cauline leaves
Figure 4. Expression analysis of FIE-silenced rosette leaves.
RT-PCR analysis detecting transcripts of: endogenous FIE; homeotic genes
AG, AGL17 and AP3; SET-domain proteins MEA and CLF; homeobox genes
BP, KNAT2, STM, KNAT6; and AS1. Ampli®cation of rRNA transcripts was
used as internal control. The number of ampli®cation cycles was optimized
for each cDNA to allow detection in the exponential range of ampli®cation as
visualized by ethidium bromide staining of the samples separated on
agarose gel electrophoresis.
ß Blackwell Publishing Ltd, The Plant Journal, (2004), 37, 707±719
It has been reported that the ectopic expression of AG
induces the development of small and curled cauline leaves
(Goodrich et al., 1997; Mizukami and Ma, 1992). Thus, the
malformed cauline leaves typical of FIE-silenced plants may
be the result of de-repression of AG. To test this hypothesis,
we generated ag-1 / /FIE-silenced mutant plants. To this
end, crosses were carried out between independent FIEsilenced lines and ag-1 heterozygote plants. ag-1 / /FIEsilenced F2 plants were identi®ed and con®rmed by RT-PCR
to be co-suppressed for the expression of both endogenous
FIE and transgenic GFP:FIE (data not shown). Unlike the FIEsilenced phenotype, in the absence of a functional AG,
ag-1 / /FIE-silenced plants displayed rosette and cauline
leaves, which were not curled (Figure 6). These results
indicate that AG overexpression indeed contributed to
712 Aviva Katz et al.
Figure 7. Similarities of gene expression pattern in rosette leaves of FIEsilenced plants and clf-2 mutant.
Total RNA was extracted from rosette leaves of Arabidopsis wild-type (wt)
Landsberg erecta (La(er)), Columbia (Col), clf-2 and FIE-silenced plants. RNA
was ampli®ed by RT-PCR using primers identifying the transcripts of AGL17,
AG, AP3, BP, KNAT2, KNAT6, STM, AS1 and MEA. Products were resolved
by agarose gel electrophoresis and stained with ethidium bromide. rRNA
ampli®cation served as an internal control.
Figure 6. Phenotypes of leaves and ¯owers of ag-1/FIE-silenced plants.
Rosette, cauline, sepals and petal leaves from wild-type Landsberg erecta
La(er), ag-1 / , FIE-silenced and ag-1 / /FIE-silenced plants are shown.
Representing samples were taken from plants of the same developmental
stage.
the curled leaf phenotype of FIE-silenced plants. As in
ag-1 / /FIE-silenced plants, the ¯owers lacked stamens
and carpels, it was not possible to determine whether
AG has a role in the development of aberrant carpel and
ovule phenotypes.
FIE and CLF control common developmental
programmes
FIE-silenced and clf mutant plants showed similar morphology of rosette and cauline leaves, ¯owers, length of
in¯orescence stem internodes, ¯owering time and ectopic
expression of AG and AP3 (Goodrich et al., 1997). RT-PCR
analysis revealed that CLF expression was not altered in
the rosette leaves of FIE-silenced plants (Figure 4), indicating that the upregulation of affected genes did not
result from the downregulation of CLF. To ®nd out
whether FIE and CLF regulate the same set of genes,
we compared the expression levels of the identi®ed derepressed genes in FIE-silenced plants and clf mutants. As
shown in Figure 7, in the rosette leaves of both FIEsilenced plants and clf-2 mutant, the expression of
AGL17, AG, AP3, KNAT2, STM and MEA was upregulated. The above results, together with the fact that
CLF is a SET domain PcG protein, strongly suggested
that CLF and FIE share common function, suppressing
expression of key developmental transcriptional regulators. BP expression was upregulated only in FIE-silenced
rosette leaves, but not in clf-2, indicating that FIE may act
via the association of proteins other than CLF to control
expression.
Interaction between FIE and CLF proteins
The common morphological characteristics of clf and FIEsilenced plants, the fact that these proteins regulate a
common set of genes, and the homology of FIE and CLF
ß Blackwell Publishing Ltd, The Plant Journal, (2004), 37, 707±719
FIE regulates homeobox gene expression in sporophyte development
713
Figure 8. FIE and CLF interact in yeast and in vitro.
(a±c) Yeast two-hybrid assays with cells expressing: GAL4BD:FIE (FIE); GAL4AD:CLF (CLF); GAL4BD:FIE with GAL4BD:CLF (FIE ‡ CLF); and GAL4BD:FIE with
GAL4AD:MEA (FIE ‡ MEA). Cells were grown on supplemented minimal media lacking leucine and tryptophan ( LT) (a) or leucine, tryptophan and histidine
( LTH) (b). Colonies grown on plate (b) were lifted on ®lters and assayed for b-galactosidase activity (c). Growth on media lacking histidine (b) and blue colour (c)
indicates interaction between FIE and either MEA (as a positive control) or CLF.
(d) Autoradiogram displaying radioactively labelled FIE that was pulled down by increasing concentrations of immobilized GST:CLF (triangle). In vitro
transcribed and translated 35S-methionine-labelled FIE (Input) was incubated with immobilized GST (20 mg) or GST-CLF protein (5, 10 and 20 mg), washed,
eluted and subjected to SDS±PAGE and autoradiography. The input FIE (Input) represents 10% of the amount of 35S-methionine-labelled FIE incubated with
immobilized proteins.
(e) Immunodetection of the in vitro transcribed FIE by polyclonal FIE antibody.
to known PcG WD40 and SET domain proteins, which
interact in Drosophila, mouse and humans (reviewed by
Berger and Gaudin, 2003; Simon and Tamkun, 2002), suggest that FIE and CLF may function together in a regulatory
complex to suppress gene expression.
To test whether FIE and CLF interact, we used yeast twohybrid assays (Figure 8). Yeast expressing either GAL4BDFIE or GAL4AD-CLF did not self-activate expression of the
reporter genes. Yeast expressing both GAL4BD-FIE and
GAL4AD-AtHD2a (Wu et al., 2000), serving as a negative
control, did not activate expression of the reporter gene
(data not shown). However, yeast expressing both
GAL4BD-FIE and GAL4AD-CLF was found to activate the
HIS3 (Figure 8b) and b-galactosidase (Figure 8c), indicating
that FIE interacts with CLF.
An in vitro pull-down assay was performed to validate
the results of the yeast two-hybrid assays (Figure 8).
To this end, CLF was expressed fused to glutathione
S-transferase (GST:CLF), and tested for its ability to bind
radioactively labelled FIE. The identity of the labelled fulllength FIE was con®rmed by its apparent size of 41 kDa
(Figure 8d), and by an anti-FIE antibody (Yadegari et al.,
2000; Figure 8e). Radioactively labelled FIE was retained
by the immobilized GST:CLF, but not by immobilized GST
alone (Figure 8d). Moreover, the quantity of retained
labelled FIE correlated with the amount of immobilized
GST:CLF used (Figure 8d), indicating the speci®city of the
interaction. Taken together, the morphological, molecular
and interaction assays strongly suggest that FIE and CLF
interact in planta to form a PcG complex, which regulates
sporophyte development.
ß Blackwell Publishing Ltd, The Plant Journal, (2004), 37, 707±719
Discussion
FIE controls sporophyte development
The FIE-silenced plants generated in the framework of this
study developed through their vegetative and reproductive
phases, to reach maturity, thus allowing us to study FIE
function and identify FIE-regulated genes and potential
partners for interaction with FIE.
FIE-silenced plants displayed a variety of morphologically abnormal phenotypes, which correlated with reduced levels of FIE transcript and protein (Figure 1). We
postulate that co-suppression occurred subsequent to
germination, allowing FIE-silenced plants to develop
beyond the seedling stage. This assumption is supported
by the ®nding that emasculated FIE-silenced plants neither
developed autonomous endosperm or seed-like structures, nor did they abort the embryo when fertilization
took place (data not shown), a characteristic of ®e mutants
(Ohad et al., 1996).
Although low levels of FIE transcripts were detected in
abnormal FIE-silenced plants (Figure 1), these levels were
probably not suf®cient to allow for normal development.
ESC, the Drosophila homologue of FIE, forms a large multiprotein PcG complex, which is highly sensitive to the initial
concentrations of its components, leading to an all-or-none
complex formation (Pirrotta and Rastelli, 1994). Similarly,
FIE is likely to be part of a multiprotein complex (Kohler
et al., 2003a; Luo et al., 1999, 2000; Spillane et al., 2000;
Yadegari et al., 2000; Figure 8). Thus, we assume that the
low levels of FIE protein in FIE-silenced plants limited the
714 Aviva Katz et al.
formation of FIE±PcG complex, resulting in the observed
pleiotropic phenotypes.
FIE is required to maintain the repression of
homeobox genes
PcG proteins maintain the repressed states of homeotic
gene expression during development (Simon and Tamkun,
2002). In Drosophila and mammals, mutations in PcG proteins lead to de-repression of their target genes, resulting in
altered developmental programmes, including homeotic
transformations. In Arabidopsis, the reduction of FIE levels
induced de-repression of key regulatory genes that control
apical dominance, ¯owering time, and leaf ¯ower and
ovule development, resulting in homeotic transformations
(Figures 4, 5 and 7). The plant PcG proteins FIE, MEA, CLF,
VRN2 and EMF2 were shown to regulate MADS-box gene
expression (Gendall et al., 2001; Goodrich et al., 1997;
Kinoshita et al., 2001; Kohler et al., 2003b), whereas in
Drosophila, the PcG complexes were found to regulate
homeobox genes. Here, we present evidence that the
PcG proteins FIE and CLF play a major role in maintaining
the repression of homeobox gene outside their appropriate
temporal and spatial expression boundaries. This may
imply that the regulatory mechanism involving the control
of homeobox genes by PcG complexes developed prior to
the evolutionary divergence of the plant and animal kingdoms, and that its functional role was conserved.
FIE-regulated homeobox genes BP, KNAT2 and STM
(Figures 4, 5 and 7), are members of the class I KNOX
genes, known to regulate shoot meristem maintenance
(Lincoln et al., 1994; Long et al., 1996; Reiser et al., 2000).
Downregulation of KNOX genes during the development of
leaf primordium is required for normal leaf morphogenesis
and persists in the mature leaf (Dockx et al., 1995; Lincoln
et al., 1994; Long et al., 1996; Semiarti et al., 2001). The
ectopic expression of BP:GUS in an FIE-silenced background (Figure 5) demonstrates that FIE controls the
expression boundaries of BP. De-repression of BP, KNAT2
and STM correlates with the FIE-silenced phenotype,
mimicking some of the phenotypes described for plants
overexpressing class I KNOX genes. STM overexpression
induces the formation of ectopic meristems (Brand et al.,
2002), and is associated with abnormal leaf development
(Brand et al., 2002; Gallois et al., 2002; Lenhard et al., 2002).
The phenotypes of FIE-silenced leaves resemble those
observed in plants overexpressing BP, resulting in lobed
leaves reduced in size and the formation of ectopic meristems on leaves (Chuck et al., 1996; Reiser et al., 2000).
KNAT2 overexpression causes curling of leaves, induces
stigmatic papillae on rosette leaves and converts ovules
into carpel-like structures (Pautot et al., 2001), resembling
the phenotypic abnormalities observed in FIE-silenced
plants. Thus, in FIE-silenced plants, ectopic expression of
BP may have contributed to serration of the leaves
(Figure 2), while the ectopic expression of KNAT2 may
contribute to leaf curl and the homeotic conversion of
ovules into carpels (Figure 3). Abnormal carpel development (Figure 2) is also consistent with the ectopic expression of AG (Figure 4), a known regulator of carpel
development. Carpel development is also regulated by
AP2 (Bowman et al., 1991) and BELL1 (BEL1) (Modrusan
et al., 1994; Robinson-Beers et al., 1992). Mutations in these
genes cause the transformation of ovules into carpel-like
structures. However, there was no change in expression
levels of BEL1 and AP2 in leaves of FIE-silenced plants
compared with the wild type (data not shown). This shows
that the conversion of ovules into carpel-like organs may
have been induced directly by KNAT2, or by other genes yet
to be determined. Putative candidates are SEPALLATA1
(SEP1), SEP2, SEP3, SHATTERPROOF1 (SHP1) and SHP2,
which are known effectors of carpel development (Favaro
et al., 2003; Pelaz et al., 2000).
AS1 and AS2 were found to suppress class I KNOX genes
in leaves (Byrne et al., 2000; Semiarti et al., 2001). As AS1
(Figure 4) and AS2 (data not shown) transcript levels
remained unchanged in the leaves of FIE-silenced plants,
it is likely that FIE represses class I KNOX genes through a
different pathway. This hypothesis is supported by the
different gene expression pro®le exhibited by FIE-silenced
plants as compared with the reported pro®les of as1 and
as2 mutants. Whereas BP, KNAT2 and KNAT6 are
expressed in as1 and as2 leaves (Byrne et al., 2000; Semiarti
et al., 2001), FIE-silenced plants express BP, KNAT2 and
STM (Figure 4). Furthermore, as1 and as2 mutants have
been shown to exhibit asymmetric leaf lobes and downward curling of leaves, and thus are phenotypically distinct
from leaves of FIE-silenced plants. Ectopic expression of
BP, KNAT2 and STM has been shown in leaves of the
double mutants ®l yab3-2 (Kumaran et al., 2002). However,
the expression of FILAMENTOUS FLOWER (FIL) and
YABBY3 (YAB3; data not shown) appears to be unchanged
in FIE-silenced plants, implying that FIE may repress the
expression of KNOX genes using different pathways.
The homeotic conversion of leaves and ¯ower organs
into carpeloid structures and the molecular evidence provided by this study indicate that another group of FIEregulated genes, including AG and AP3, belongs to the
MADS-box gene family, which is consistent with the ®ndings of other studies (Yadegari et al., 2000). The ectopic
expression of homeobox and MADS-box genes described
in this work establishes that in wild-type plants, FIEmediated repression occurs throughout the entire vegetative phase of plant development.
The C-class organ identity gene, AG, is known to regulate
stamen and carpel development (Bowman et al., 1989;
Yanofsky et al., 1990), as well as ¯oral meristem determinacy (Busch et al., 1999; Mizukami and Ma, 1995; Okamuro
ß Blackwell Publishing Ltd, The Plant Journal, (2004), 37, 707±719
FIE regulates homeobox gene expression in sporophyte development
et al., 1996). FIE-silenced plants, which ectopically
expressed AG, resembled partially AG-overexpressing
plants (Figures 2, 4 and 6). Accordingly, the curled leaf
phenotype of FIE-silenced plants was suppressed in the
ag-1 background (Figure 6). These results demonstrate
that AG overexpression plays a role in the curled leaf
phenotype.
KNAT2 was ectopically expressed in FIE-silenced rosette
leaves (Figure 4). Ectopic expression of KNAT2 was shown
to induce expression of AG (Pautot et al., 2001). No changes
in the expression levels of other AG regulators, such as
WUS (Lenhard et al., 2001), AP2 (Bowman et al., 1991;
Drews et al., 1991; Kunst et al., 1989; Modrusan et al.,
1994), or ANT (Krizek et al., 2000) were detected (data not
shown). Thus, AG-elevated expression either may have
resulted from the direct de-repression of AG in the absence
of the FIE±PcG complex, or may have been activated by
KNAT2. Similarly, CLF may regulate AG expression via
KNAT2, as indicated from the upregulation of KNAT2 in
clf mutants (Figure 7).
It is interesting to note the phenotypic resemblance
between FIE-silenced elongated ovules and those observed
in petunia, which resulted from co-suppression of Fbp7 and
Fbp11 MADS box-like genes that are regulators of ovule
identity (Angenent et al., 1995). However, in FIE-silenced
plants, we detect no change in expression level of SEEDSTICK (STK; previously AGL11), which is considered to be
the Arabidopsis orthologue of Fbp7 and Fbp11 (Becker and
Theissen, 2003). Other members of the MADS-box gene
family, AG and AGL17, were upregulated (Figures 4 and 7).
As in wild-type plants AGL17 expression is restricted to the
roots (Burgeff et al., 2002; Rounsley et al., 1995), the importance of FIE repression of AGL17 expression in the shoot
remains to be determined.
FIE may form alternative functional complexes with
different PcG SET domain proteins
FIE and CLF interaction, demonstrated by yeast two-hybrid
and in vitro binding assays (Figure 8), implies that both
proteins may interact in vivo to form a PcG complex. Moreover, the leaf and ¯ower phenotypes of FIE-silenced plants
resemble those of clf mutants (Goodrich et al., 1997), while
both proteins regulate a common set of transcription factors, members of the homeobox and MADS-box gene
families (Figure 7). Thus, FIE interacts with different members of the SET domain protein family such as MEA (Luo
et al., 1999, 2000; Spillane et al., 2000; Yadegari et al., 2000)
and CLF (Figure 8), possibly allowing the formation of
various PcG complexes, which regulate different developmental programmes. Supporting the above is the observation that BP is de-repressed in FIE-silenced plants but not in
clf-2 mutants, indicating that FIE may associate with different PcG proteins other than CLF, in order to mediate represß Blackwell Publishing Ltd, The Plant Journal, (2004), 37, 707±719
715
sion of BP. The dynamics of PcG's organization during
developmental programmes has been shown in Drosophila
(Furuyama et al., 2003).
De-repression of MEA was observed in rosette leaves of
both clf and FIE-silenced plants (Figures 4 and 7). These
results indicate that in wild-type plants, the PcG complex
containing both FIE and CLF downregulates MEA expression in the sporophyte. Such regulation of MEA expression
may lead to the formation of a FIE±CLF complex and suppress the formation of FIE±MEA complex in the sporophyte.
DEMETER (DME), another known regulator of MEA,
causes ectopic expression of MEA in rosette leaves when
overexpressed (Choi et al., 2002). However, it appears that
DME did not mediate the upregulation of MEA in FIEsilenced plants and clf-2 mutants because DME expression
levels were not changed in these plants (data not shown).
Potential partners of Arabidopsis PcG complexes
FIE PcG complexes maintain the restricted pattern of various homeobox and MADS-box gene expression, thus
mediating the control of reproductive and vegetative programmes. The results of this study outline the unique
ability of FIE in plants to interact with different PcG SET
domain proteins. Genetic and biochemical studies may
help to determine potential constituents of these complexes. The Drosophila ESC±Ez complex was shown to
contain p55, an Rb-binding protein, and Su(z)12, a zinc
®nger protein (Luo et al., 1999; Muller et al., 2002; Ng et al.,
2000; Tie et al., 2001). In Arabidopsis, recent ®ndings show
that the FIE±MEA complex in seeds contains MSI (Kohler
et al., 2003a), a homologue of p55 (Ach et al., 1997), which
extends the structural conservation between the plant and
Drosophila's PcG complexes. FIS2, a zinc ®nger protein,
was suggested to be part of this complex because of the
similarities between the mutant phenotypes of ®s2 and
those of ®e, mea and msi1, initiating autonomous endosperm development. FIE, MEA and FIS2 repress the expression of PHE, which further supports their participation in a
common PcG complex (Kohler et al., 2003b). The proposed
FIE±CLF complex may also harbour EMF2, a homologue of
Su(z)12, as a zinc ®nger member of the complex. This
model is supported by phenotypic similarities among the
various mutants. For example, ®e (Kinoshita et al., 2001), clf
(Goodrich et al., 1997) and emf2 (Sung et al., 1992) mutants
all display early ¯owering, and clf (Goodrich et al., 1997),
co-suppressed FIE and co-suppressed EMF2 (Goodrich
et al., 1997) display the curled leaf phenotype. Genetic
analysis (Haung and Yang, 1998) reveals that EMF2 independently controls ¯owering time and rosette leaf formation, thus further supporting the above notion that EMF2
may function together with FIE and CLF to regulate the
vegetative phase. The FIE±CLF±EMF2 complex proposed
here possibly contains MSI1, similar to the putative
716 Aviva Katz et al.
FIE±MEA±FIS2±MSI1 complex in seeds, just as the MSI1
homologue was found to be part of the Drosophila PcG
complex. This is further supported by the similarity in
phenotype of FIE and MSI co-suppressed plants (Hennig
et al., 2003), displaying reduced apical dominance, curled
leaves, and aberrant ¯owers and ovules.
Experimental procedures
Plant material and growth conditions
Arabidopsis thaliana ecotype Columbia glabra served as the wild
type. clf-2 and ag-1 mutants were obtained from the Arabidopsis
Biological Resource Center (Ohio State University, Columbus, OH,
USA). Plants were grown at 228C under 16-h light and 8-h dark
photoperiods in Percivall growth chambers.
Plant morphology
Plant morphology was documented using a SV11-stereomicroscope and Axioplan-2 microscope (Carl Zeiss Inc., Gottingen,
Germany) and photographed with a Nikon-coolpix 950 (Tokyo,
Japan) camera. Image processing was performed with PHOTOSHOP 7.0.2 (Adobe Systems, San Jose, CA, USA).
Generation of transgenic plants
p35S-GFP:FIE was constructed as a translational fusion between
GFP(S65T) and FIE cDNA at N-terminus. First, FIE cDNA was
inserted between the CaMV-35S promoter and the polyadenylation sequence, using SalI and XmaI sites of pMD1 binary vector to
create pMDI-FIE. pMD1 is a derivative of the pBI121 vector (Clontech, Palo Alto, CA, USA) in which the GUS gene was replaced by a
multiple cloning site. In the second stage, the GFP was ampli®ed
by PCR, with a forward primer harbouring both BamHI and NcoI
sites (50 -GCGGATCCATGGTGAGC-30 ), and a reverse primer, which
abolished the stop codon and contained a BamHI site (50 GCGGATCCCTTGTACAGCTCGTCC-30 ). The PCR product was
digested with BamHI and subcloned into the pMDI-FIE vector,
resulting in the pMD1-GFP:FIE. A 15-bp spacer encoding a KGSPG
amino acid sequence was added between the FIE- and GFP-coding
sequences because of the cloning procedure. All constructs were
veri®ed by sequencing. The binary vector was transformed into
Agrobacterium GV3101 pMP90 (Koncz and Schell, 1986). Transformation of Arabidopsis was performed by the ¯oral dip method
(Clough and Bent, 1998). Sixty independent kanamycin-resistant
T1 plants were obtained and con®rmed to contain the desired
insert by PCR.
Total RNA isolation and RT-PCR analyses
Sample tissue was collected from Arabidopsis plants and immediately frozen in liquid nitrogen. Total RNA was prepared using an
SV total RNA isolation kit (Promega, Madison, WI, USA). For
reverse transcription, 12 mg of total RNA was incubated for 1 h
at 428C with 300 units of Moloney Murine leukaemia virus H
superscript reverse transcriptase (Invitrogen Corp., Carlsbad,
CA, USA) in 70 ml reaction mixture containing 215 pmol of 21oligo
(dT), appropriate buffer, 10 mM DTT, 0.6 mM dNTPs (Roche,
Mannheim, Germany) and primer 3404 (50 -ACATCTAAGGGCAT-
CACAGAC-30 ) for priming ribosomal RNAs. cDNAs equalled to
one-seventh of the total RNA in the reverse transcription reaction
mixtures were ampli®ed with ExTaq polymerase (Takara, Otsu,
Japan) in 50 ml of reaction mixtures. 18S ribosomal RNA served as
an internal control for monitoring reaction ef®ciency and to assure
that the initial amounts of cDNA were equal. 18S ribosomal RNA
was ampli®ed with (50 -TGCAGTTAAAAAGCTCGTAGTTG-30 ) and
(50 -ACATCTAAGGGCATCACAGAC-30 ) primers. The GFP:FIE transgene was ampli®ed with a GFP forward primer (50 -GCGGATCCATGGTGAGCAAGG-30 ) and an FIE reverse primer (50 -CCGCTCGAGCTACTTGGTAATCACGTC-30 ). Endogenous FIE cDNA was ampli®ed with a forward primer (50 -GATTGTCGACTCGAGATGTCGAAGATAACC-30 ) and a reverse primer from the 30 untranslated region
(50 -CTCCAGAAAGGGTATACACTG-30 ). Expression of MADS-box
and homeobox genes was examined using a self-developed RTPCR ampli®cation kit (Patent case # 133684, Tel Aviv, Israel). Other
speci®c cDNA targets were ampli®ed using the following primer
sets: MEA (50 -GCAGGACTATGGTTTGGATG-30 ) and (50 GATCAGAGGATTGGTCTATTTGC-30 ); AP3 (50 -ATGGCGAGAGGGAAGATCCAG-30 ) and (50 -GATGGCACCAGCAAACCTTTTAG-30 ); AGL17 (50 CGGGATCCTAGAACGCACCAAGATCTAAAGG-30 ) and (50 -CGGCTCGAGTTAGCTGTTTGAAGATGTCTTATAATGG-30 ); CLF (50 -ATGGCGTCAGAAGCTTCGC-30 ) and (50 -CTTCCAGACTTGAGAAGCG-30 );
AG (50 -CTAGGAGGAGATTCCTCTCC-30 ) and (50 -CTAACTGGAGAGCGGTTTGG-30 ); STM (50 -GTGCTCCTGCCTATTCTCTAATG-30 ) and
(50 -CTATCCTCAGTTGTGGATCTAC-30 ); BP (50 -GGGTATGGAAGAATACCAGC-30 ) and (50 -TATGGACCGAGACGATAAG-30 ); KNAT2
(50 -GAAGAGATTCAGCGAGAGAACC-30 ) and (50 -GAATCGTCCATCATATCAAACGGCATG-30 ); KNAT6 (50 -GATGATGTCACCGGAGAGTCTC-30 ) and (50 -GACTCGACACCAGTACATAGGTTC-30 ); AS1 (50 ATGAAAGAGAGACAACGTTGGAG-30 ) and (50 -GAACACACTCTCGCTACTC-30 ); and AS2 (50 -CTCTCAATTTTCAATGGCGGCTTTGTG-30 ) and (50 -CTCAAGACGGATCAACAGTACG-30 ). The annealing
temperature used for all sets of primers was 608C.
Yeast two-hybrid clones and assays
Full-length FIE cDNA was subcloned into the pBI880 vector containing the GAL4-binding domain. The full-length CLF cDNA was
ampli®ed by PCR and subcloned into the pBI771 vector containing
the GAL4 activation domain (Kohalmi et al., 1997). All clones were
veri®ed by sequencing. Yeast two-hybrid assays were carried out
as previously described by Yadegari et al. (2000).
Detection of protein±protein interaction by
pull-down assay
Pull-down assays were performed according to Yadegari et al.
(2000). 35S-radioactively labelled FIE was produced as previously
described by Ohad et al. (1999), by expressing FIE cDNA in the
pCITE vector (Novagen, Darmstadt, Germany), allowing coupled
transcription±translation of the FIE protein radioactively labelled
by 35S-methionine. CLF full-length cDNA was cloned by RT-PCR,
and subsequently subcloned into the pGEX-4T-1 vector (Amersham Pharmacia Biotechnology, Piscataway, NJ, USA), allowing
expression and puri®cation of the CLF-GST fusion protein. Puri®ed
GST:CLF (5, 10 and 20 mg) or control GST (20 mg) proteins were
immobilized on glutathione-agarose beads (Sigma, St Louis, MO,
USA), washed with 20 mM Tris±HCl buffer (pH 8.0) containing
100 mM NaCl, 1 mM EDTA and 0.5% Nonidet-P40, and re-suspended in 200 ml of the same buffer to which the labelled FIE
was added and incubated for 2 h at 48C. The beads were washed
four times with the same buffer, re-suspended in sample buffer
ß Blackwell Publishing Ltd, The Plant Journal, (2004), 37, 707±719
FIE regulates homeobox gene expression in sporophyte development
and resolved by SDS±PAGE. The proteins were transferred to
polyvinylidene ¯uoride (PVDF) membranes (Millipore, Bedford,
MA, USA). Labelled proteins were detected by autoradiography
after exposing the membranes to X-ray BIOMAX MS ®lm (Kodak,
Rochester, NY, USA).
Total Arabidopsis protein extraction and
Western blotting
Rosette leaves were harvested and ground to powder in liquid
nitrogen. The tissue was further ground with protein extraction
buffer (100 mM Tris (pH 7.2), 10% sucrose, 5 mM MgCl2, 5 mM
EDTA, 40 mM b-mercaptoethanol and protease inhibitor cocktail
(Roche, Mannheim, Germany) in a 1 : 1 ratio to the tissue weight.
The extract was then centrifuged, and the supernatant was used
for Western blot analysis. Total protein concentration was determined by Bradford assay (Bradford, 1976), and equal amounts of
protein were resolved on a 10% SDS±PAGE and transferred onto
nitrocellulose membranes. To detect FIE protein, membranes were
incubated with rabbit anti-FIE polyclonal antibody (Yadegari et al.,
2000) in a 1 : 500 dilution, and then washed and incubated with
blotting-grade horseradish peroxidase-conjugated goat-antirabbit
secondary antibody (Jackson Immuno Research, West Grove, PN,
USA). Detection was carried out using paracoumaric acid and
luminol (Sigma, St Louis, MO, USA), according to the manufacturer's instructions.
Acknowledgements
We wish to thank Dr Ben-Tzion Vider for his assistance in developing a PCR-based kit to examine the expression of gene families;
Dr Naomi Ori for providing the BP:GUS seeds, The Arabidopsis
Biological Resource Center for providing ag-1 and clf-2 seeds; and
Daniella Bar-El, Marina Kalis and Noa Zecharia for technical assistance. We thank Dr S. Yalovsky and Dr N. Ori for their critical
reading of the manuscript. O.H. was supported by the Ministry of
Science Eshkol Fellowship, Israel. This research was supported by
Israel Science Foundation Grant No-503-00, and by BARD, the
United States±Israel Binational Agricultural Research and Development Fund, Grant No. IS-3158-99C.
Supplementary Material
The following material is available from http://www.blackwell
publishing.com/products/journals/suppmat/TPJ/TPJ1996/TPJ1996sm.
htm
Figure S1. Expression analysis of different FIE-silenced plants
tissues.
Ethidium bromide-stained gels detecting FIE, AG, AGL17, MEA,
AP3 and BP transcripts by RT-PCR analysis. Total RNA was extracted from rosette leaves, cauline leaves and sepals of wild-type and
FIE-silenced plants exhibiting abnormal phenotypes. Ampli®cation of ubiquitin (UBI) transcripts was used as internal controls.
References
Ach, R.A., Taranto, P. and Gruissem, W. (1997) A conserved family
of WD-40 proteins binds to the retinoblastoma protein in both
plants and animals. Plant Cell, 9, 1595±1606.
Angenent, G.C., Franken, J., Busscher, M., van Dijken, A., van
Went, J.L., Dons, H.J. and van Tunen, A.J. (1995) A novel class
ß Blackwell Publishing Ltd, The Plant Journal, (2004), 37, 707±719
717
of MADS-box genes is involved in ovule development in petunia. Plant Cell, 7, 1569±1582.
Becker, A. and Theissen, G. (2003) The major clades of MADS-box
genes and their role in the development and evolution of ¯owering plants. Mol. Phylogenet. Evol. 29, 464±489.
Berger, F. and Gaudin, V. (2003) Chromatin dynamics and Arabidopsis development. Chromosome Res. 11, 277±304.
Bowman, J.L., Smyth, D.R. and Meyerowitz, E.M. (1989) Genes
directing ¯ower development in Arabidopsis. Plant Cell, 1,
37±52.
Bowman, J.L., Smyth, D.R. and Meyerowitz, E.M. (1991) Genetic
interactions among ¯oral homeotic genes of Arabidopsis. Development, 112, 1±20.
Bowman, J.L., Sakai, H., Jack, T., Weigel, D., Mayer, U. and
Meyerowitz, E.M. (1992) SUPERMAN, a regulator of ¯oral
homeotic genes in Arabidopsis. Development, 114, 599±615.
Bradford, M.M. (1976) A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle
of protein-dye binding. Anal. Biochem. 72, 248±254.
Brand, U., Grunewald, M., Hobe, M. and Simon, R. (2002) Regulation of CLV3 expression by two homeobox genes in Arabidopsis.
Plant Physiol. 129, 565±575.
Burgeff, C., Liljegren, S.J., Tapia-Lopez, R., Yanofsky, M.F. and
Alvarez-Buylla, E.R. (2002) MADS-box gene expression in lateral
primordia, meristems and differentiated tissues of Arabidopsis
thaliana roots. Planta, 214, 365±372.
Busch, M.A., Bomblies, K. and Weigel, D. (1999) Activation of a
¯oral homeotic gene in Arabidopsis. Science, 285, 585±587.
Byrne, M.E., Barley, R., Curtis, M., Arroyo, J.M., Dunham, M.,
Hudson, A. and Martienssen, R.A. (2000) ASYMMETRIC
LEAVES1 mediates leaf patterning and stem cell function in
Arabidopsis. Nature, 408, 967±971.
Byrne, M.E., Simorowski, J. and Martienssen, R.A. (2002) ASYMMETRIC LEAVES1 reveals KNOX gene redundancy in Arabidopsis. Development, 129, 1957±1965.
Chaudhury, A.M., Lavithis, M., Taylor, P.E., Craig, S.M.B.S., Singer,
E.R., Knox, R.B., Dennis, E.S. and Lavithis, M. (1994) Genetic
control of male fertility in Arabidopsis thaliana: structural analysis of premeiotic developmental mutants. Sex. Plant Reprod. 7,
17±28.
Chaudhury, A.M., Craig, S., Dennis, E. and Peacock, W. (1998)
Ovule and embryo development, apomixis and fertilization.
Curr. Opin. Plant. Biol. 1, 26±31.
Choi, Y., Gehring, M., Johnson, L., Hannon, M., Harada, J.J.,
Goldberg, R.B., Jacobsen, S.E. and Fischer, R.L. (2002)
DEMETER, a DNA glycosylase domain protein, is required for
endosperm gene imprinting and seed viability in Arabidopsis.
Cell, 110, 33±42.
Chuck, G., Lincoln, C. and Hake, S. (1996) KNAT1 induces lobed
leaves with ectopic meristems when overexpressed in Arabidopsis. Plant Cell, 8, 1277±1289.
Clough, S.J. and Bent, A.F. (1998) Floral dip: a simpli®ed method
for Agrobacterium-mediated transformation of Arabidopsis
thaliana. Plant J. 16, 735±743.
Denli, A.M. and Hannon, G.J. (2003) RNAi: an ever-growing puzzle.
Trends Biochem. Sci. 28, 196±201.
Dockx, J., Quaedvlieg, N., Keultjes, G., Kock, P., Weisbeek, P. and
Smeekens, S. (1995) The homeobox gene ATK1 of Arabidopsis
thaliana is expressed in the shoot apex of the seedling and in
¯owers and in¯orescence stems of mature plants. Plant Mol.
Biol. 28, 723±737.
Drews, G.N., Bowman, J.L. and Meyerowitz, E.M. (1991) Negative
regulation of the Arabidopsis homeotic gene AGAMOUS by the
APETALA2 product. Cell, 65, 991±1002.
718 Aviva Katz et al.
Favaro, R., Pinyopich, A., Battaglia, R., Kooiker, M., Borghi, L.,
Ditta, G., Yanofsky, M.F., Kater, M.M. and Colombo, L. (2003)
MADS-box protein complexes control carpel and ovule development in Arabidopsis. Plant Cell, 15, 2603±2611.
Furuyama, T., Tie, F. and Harte, P.J. (2003) Polycomb group
proteins ESC and E(Z) are present in multiple distinct complexes
that undergo dynamic changes during development. Genesis,
35, 114±124.
Gallois, J.L., Woodward, C., Reddy, G.V. and Sablowski, R. (2002)
Combined SHOOTMERISTEMLESS and WUSCHEL trigger ectopic
organogenesis in Arabidopsis. Development, 129, 3207±3217.
Gendall, A.R., Levy, Y.Y., Wilson, A. and Dean, C. (2001) The
VERNALIZATION 2 gene mediates the epigenetic regulation of
vernalization in Arabidopsis. Cell, 107, 525±535.
Goodrich, J. and Tweedie, S. (2002) Remembrance of things past:
chromatin remodeling in plant development. Annu. Rev. Cell
Dev. Biol. 18, 707±746.
Goodrich, J., Puangsomlee, P., Martin, M., Long, D., Meyerowitz,
E.M. and Coupland, G. (1997) A Polycomb-group gene regulates
homeotic gene expression in Arabidopsis. Nature, 386, 44±51.
Haung, M.D. and Yang, C.H. (1998) EMF genes interact with late¯owering genes to regulate Arabidopsis shoot development.
Plant Cell Physiol. 39, 382±393.
Hennig, L., Taranto, P., Walser, M., Schonrock, N. and Gruissem,
W. (2003) Arabidopsis MSI1 is required for epigenetic
maintenance of reproductive development. Development, 130,
2555±2565.
Hsieh, T.-F., Hakim, O., Ohad, N. and Fischer, R.L. (2003) From ¯our
to ¯ower: how Polycomb group proteins in¯uence multiple
aspects of plant development. Trends Plant Sci. 8, 439±445.
Jones, C.A., Ng, J., Peterson, A.J., Morgan, K., Simon, J. and
Jones, R.S. (1998) The Drosophila ESC and E(z) proteins are
direct partners in polycomb group-mediated repression. Mol.
Cell Biol. 18, 2825±2834.
Kinoshita, T., Harada, J.J., Goldberg, R.B. and Fischer, R.L. (2001)
Polycomb repression of ¯owering during early plant development. Proc. Natl. Acad. Sci. USA, 98, 14156±14161.
Kohalmi, S.E., Nowak, J. and Crosby, W.I. (1997) A practical guide
to using the yeast 2-hybrid system. In Differentially Expressed
Genes in Plants (Hansen, E. and Harper, G., eds.). London: Taylor
and Francis, pp. 63±82.
Kohler, C. and Grossniklaus, U. (2002) Epigenetics: the ¯owers that
come in from the cold. Curr. Biol. 12, R129±R131.
Kohler, C., Hennig, L., Bouveret, R., Gheyselinck, J., Grossniklaus,
U. and Gruissem, W. (2003a) Arabidopsis MSI1 is a component
of the MEA/FIE Polycomb group complex and required for seed
development. EMBO J. 22, 4804±4814.
Kohler, C., Hennig, L., Spillane, C., Pien, S., Gruissem, W. and
Grossniklaus, U. (2003b) The Polycomb-group protein MEDEA
regulates seed development by controlling expression of the
MADS-box gene PHERES1. Genes Dev. 17, 1540±1553.
Koncz, C. and Schell, J. (1986) The promoter of TL-DNA gene 5
controls the tissue speci®c expression of chimeric genes carried
by a novel type of Agrobacterium binary vector. Mol. Gen.
Genet. 204, 383±396.
Krizek, B.A., Prost, V. and Macias, A. (2000) AINTEGUMENTA
promotes petal identity and acts as a negative regulator of
AGAMOUS. Plant Cell, 12, 1357±1366.
Kumaran, M.K., Bowman, J.L. and Sundaresan, V. (2002) YABBY
polarity genes mediate the repression of KNOX homeobox
genes in Arabidopsis. Plant Cell, 14, 2761±2770.
Kunst, L., Klenz, J.E., Martinez-Zapater, J. and Haughn, G.W.
(1989) AP2 gene determines the identity of perianth organs in
¯owers of Arabidopsis thaliana. Plant Cell, 1, 1195±1208.
Lenhard, M., Bohnert, A., Jurgens, G. and Laux, T. (2001) Termination of stem cell maintenance in Arabidopsis ¯oral meristems
by interactions between WUSCHEL and AGAMOUS. Cell, 105,
805±814.
Lenhard, M., Jurgens, G. and Laux, T. (2002) The WUSCHEL and
SHOOTMERISTEMLESS genes ful®ll complementary roles in
Arabidopsis shoot meristem regulation. Development, 129,
3195±3206.
Lincoln, C., Long, J., Yamaguchi, J., Serikawa, K. and Hake, S.
(1994) A knotted1-like homeobox gene in Arabidopsis is
expressed in the vegetative meristem and dramatically alters
leaf morphology when overexpressed in transgenic plants. Plant
Cell, 6, 1859±1876.
Lohmann, J.U., Hong, R.L., Hobe, M., Busch, M.A., Parcy, F.,
Simon, R. and Weigel, D. (2001) A molecular link between stem
cell regulation and ¯oral patterning in Arabidopsis. Cell, 105,
793±803.
Long, J.A., Moan, E.I., Medford, J.I. and Barton, M.K. (1996) A
member of the KNOTTED class of homeodomain proteins
encoded by the STM gene of Arabidopsis. Nature, 379, 66±69.
Luo, M., Bilodeau, P., Koltunow, A., Dennis, E.S., Peacock, W.J.
and Chaudhury, A.M. (1999) Genes controlling fertilization-independent seed development in Arabidopsis thaliana. Proc. Natl.
Acad. Sci. USA, 96, 296±301.
Luo, M., Bilodeau, P., Dennis, E.S., Peacock, W.J. and Chaudhury,
A. (2000) Expression and parent-of-origin effects for FIS2, MEA,
and FIE in the endosperm and embryo of developing Arabidopsis seeds. Proc. Natl. Acad. Sci. USA, 97, 10637±10642.
Mizukami, Y. and Ma, H. (1992) Ectopic expression of the ¯oral
homeotic gene AGAMOUS in transgenic Arabidopsis plants
alters ¯oral organ identity. Cell, 71, 119±131.
Mizukami, Y. and Ma, H. (1995) Separation of AG function in ¯oral
meristem determinacy from that in reproductive organ identity
by expressing antisense AG RNA. Plant Mol. Biol. 28, 767±784.
Modrusan, Z., Reiser, L., Feldmann, K.A., Fischer, R.L. and Haughn,
G.W. (1994) Homeotic transformation of ovules into carpel-like
structures in Arabidopsis. Plant Cell, 6, 333±349.
Moon, Y.H., Chen, L., Pan, R.L., Chang, H.S., Zhu, T., Maffeo, D.M.
and Sung, Z.R. (2003) EMF genes maintain vegetative development by repressing the ¯ower program in Arabidopsis. Plant
Cell, 15, 681±693.
Muller, J., Hart, C.M., Francis, N.J., Vargas, M.L., Sengupta, A.,
Wild, B., Miller, E.L., O'Connor, M.B., Kingston, R.E. and Simon,
J.A. (2002) Histone methyltransferase activity of a Drosophila
Polycomb group repressor complex. Cell, 111, 197±208.
Ng, J., Hart, C.M., Morgan, K. and Simon, J.A. (2000) A Drosophila
ESC-E(Z) protein complex is distinct from other polycomb group
complexes and contains covalently modi®ed ESC. Mol. Cell Biol.
20, 3069±3078.
Ohad, N., Margossian, L., Hsu, Y.C., Williams, C., Repetti, P.
and Fischer, R.L. (1996) A mutation that allows endosperm
development without fertilization. Proc. Natl Acad. Sci. USA,
93, 5319±5324.
Ohad, N., Yadegari, R., Margossian, L., Hannon, M., Michaeli, D.,
Harada, J.J., Goldberg, R.B. and Fischer, R.L. (1999) Mutations in
FIE, a WD polycomb group gene, allow endosperm development
without fertilization. Plant Cell, 11, 407±416.
Okamuro, J.K., den Boer, B.G., Lotys-Prass, C., Szeto, W. and
Jofuku, K.D. (1996) Flowers into shoots: photo and hormonal
control of a meristem identity switch in Arabidopsis. Proc. Natl.
Acad. Sci. USA, 93, 13831±13836.
Ori, N., Eshed, Y., Chuck, G., Bowman, J.L. and Hake, S. (2000)
Mechanisms that control knox gene expression in the Arabidopsis shoot. Development, 127, 5523±5532.
ß Blackwell Publishing Ltd, The Plant Journal, (2004), 37, 707±719
FIE regulates homeobox gene expression in sporophyte development
Pautot, V., Dockx, J., Hamant, O., Kronenberger, J., Grandjean, O.,
Jublot, D. and Traas, J. (2001) KNAT2: evidence for a link
between knotted-like genes and carpel development. Plant Cell,
13, 1719±1734.
Pelaz, S., Ditta, G.S., Baumann, E., Wisman, E. and Yanofsky, M.F.
(2000) B and C ¯oral organ identity functions require SEPALLATA MADS-box genes. Nature, 405, 200±203.
Pirrotta, V. and Rastelli, L. (1994) White gene expression, repressive chromatin domains and homeotic gene regulation in Drosophila. Bioessays, 16, 549±556.
Reiser, L., Sanchez-Baracaldo, P. and Hake, S. (2000) Knots in the
family tree: evolutionary relationships and functions of knox
homeobox genes. Plant Mol. Biol. 42, 151±166.
Riechmann, J.L. and Meyerowitz, E.M. (1977) MADS domain proteins in plant development. Biol. Chem. 378, 1079±1101.
Robinson-Beers, K., Pruitt, R.E. and Gasser, C.S. (1992) Ovule
development in wild-type Arabidopsis and two female-sterile
mutants. Plant Cell, 4, 1237±1249.
Rounsley, S.D., Ditta, G.S. and Yanofsky, M.F. (1995) Diverse roles
for MADS-box genes in Arabidopsis development. Plant Cell, 7,
1259±1269.
Sakai, H., Medrano, L.J. and Meyerowitz, E.M. (1995) Role of
SUPERMAN in maintaining Arabidopsis ¯oral whorl boundaries. Nature, 378, 199±203.
Semiarti, E., Ueno, Y., Tsukaya, H., Iwakawa, H., Machida, C.
and Machida, Y. (2001) The ASYMMETRIC LEAVES2 gene
of Arabidopsis thaliana regulates formation of a symmetric
lamina, establishment of venation and repression of meristem-related homeobox genes in leaves. Development, 128,
1771±1783.
Sewalt, R.G., van der Vlag, J., Gunster, M.J., Hamer, K.M., den
Blaauwen, J.L., Satijn, D.P., Hendrix, T., van Driel, R. and Otte,
A.P. (1998) Characterization of interactions between the mammalian polycomb-group proteins Enx1/EZH2 and EED suggests
the existence of different mammalian polycomb-group protein
complexes. Mol. Cell Biol. 18, 3586±3595.
Simon, J.A. and Tamkun, J.W. (2002) Programming off and
on states in chromatin: mechanisms of Polycomb and
ß Blackwell Publishing Ltd, The Plant Journal, (2004), 37, 707±719
719
trithorax group complexes. Curr. Opin. Genet. Dev. 12, 210±
218.
Spillane, C., MacDougall, C., Stock, C., Kohler, C., Vielle-Calzada,
J.P., Nunes, S.M., Grossniklaus, U. and Goodrich, J. (2000)
Interaction of the Arabidopsis polycomb group proteins FIE
and MEA mediates their common phenotypes. Curr. Biol. 10,
1535±1538.
Sung, Z.R., Belachew, A., Shunong, B. and Bertrand-Garcia, R.
(1992) EMF, an Arabidopsis gene required for vegetative shoot
development. Science, 258, 1645±1647.
Sung, Z.R., Chen, L., Moon, Y.H. and Lertpiriyapong, K. (2003)
Mechanisms of ¯oral repression in Arabidopsis. Curr. Opin.
Plant. Biol. 6, 29±35.
Szweykowska-Kulinska, Z., Jarmolowski, A. and Figlerowicz, M.
(2003) RNA interference and its role in the regulation of eucaryotic gene expression. Acta Biochim. Pol. 50, 217±229.
Tie, F., Furuyama, T. and Harte, P.J. (1998) The Drosophila Polycomb Group proteins ESC and E(Z) bind directly to each other
and co-localize at multiple chromosomal sites. Development,
125, 3483±3496.
Tie, F., Furuyama, T., Prasad-Sinha, J., Jane, E. and Harte, P.J.
(2001) The Drosophila Polycomb group proteins ESC and
E(Z) are present in a complex containing the histone-binding
protein p55 and the histone deacetylase RPD3. Development,
128, 275±286.
Wagner, D. (2003) Chromatin regulation of plant development.
Curr. Opin. Plant. Biol. 6, 20±28.
Wu, K., Tian, L., Malik, K., Brown, D. and Miki, B. (2000) Functional
analysis of HD2 histone deacetylase homologues in Arabidopsis
thaliana. Plant J. 22, 19±27.
Yadegari, R., Kinoshita, T., Lotan, O. et al. (2000) Mutations in the
FIE and MEA genes that encode interacting polycomb proteins
cause parent-of-origin effects on seed development by distinct
mechanisms. Plant Cell, 12, 2367±2382.
Yanofsky, M.F., Ma, H., Bowman, J.L., Drews, G.N., Feldmann, K.A.
and Meyerowitz, E.M. (1990) The protein encoded by the Arabidopsis homeotic gene AGAMOUS resembles transcription factors. Nature, 346, 35±39.