Department of Botany, Graduate School of Science, Kyoto University, Kyoto, 606-8502, Japan
Author for correspondence (e-mail:
kiyo{at}ok-lab.bot.kyoto-u.ac.jp)
Accepted 21 October 2003
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SUMMARY |
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Key words: Arabidopsis thaliana, Petal development, RABBIT EARS (RBE), Zinc finger protein
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Introduction |
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The identity of floral organs is controlled by three classes of homeotic
genes, termed ABC genes, which encode MADS-box transcription factors
(Goto and Meyerowitz, 1994;
Jack et al., 1992
;
Mandel et al., 1992
;
Yanofsky et al., 1990
).
According to the present model, the ABC genes determine the fate of floral
organs, which depends on the whorl the organ primordia are formed in
(Bowman et al., 1991
;
Coen and Meyerowitz, 1991
;
Meyerowitz et al., 1991
;
Weigel and Meyerowitz, 1994
).
This idea is consistent with findings that ABC genes are expressed in a
ring-shaped region that corresponds to hypothetical whorls. In addition to the
ABC homeotic genes, another class of MADS-box genes, SEPALLATA1, 2
and 3 (SEP1, 2 and 3) has been reported
(Pelaz et al., 2000
). In a
sep1 sep2 sep3 triple mutant, petals and stamens are transformed into
sepalloid organs, and the innermost whorl is replaced by a new flower that
repeats this same phenotype. Activities of SEP2 or SEP3 with
other floral homeotic genes can convert vegetative leaves into floral organs
(Honma and Goto, 2001
;
Pelaz et al., 2001a
;
Pelaz et al., 2001b
). It is
unlikely, however, the floral homeotic genes define the size and position of
whorls, because the mutations in these genes do not generally affect the size
and position of floral organ primordia. For example, in pistillata
(pi), one of a class B mutant, early development of organ primordia
is indistinguishable from that of wild type
(Hill and Lord, 1989
). This
indicates that the concentric regions have been defined spatially before these
genes start to be expressed.
Petals are the most conspicuous organs in a flower because their colors and
shapes vary widely among plant species. According to the ABC model, petal
identity is established in the second whorl by class A, class B and
SEP genes. Because these genes are expressed in a ring-like pattern,
other factors are required for the proper arrangement of petal primordia in a
floral meristem. Floral organ numbers increase in clavata
(clv) mutants because of the enlarged floral meristem
(Clark et al., 1993;
Clark et al., 1995
;
Kayes and Clark, 1998
). In
perianthia (pan), the number of sepals, petals and stamens
changes to five, which suggests that PAN is required for the
establishment of the tetramerous structure of a flower
(Running and Meyerowitz,
1996
). Thus, these genes control the number of floral organ
primordia.
After petal primordia form, they follow the process of cell proliferation
and cell specialization to develop to mature petals. However, the mechanisms
remain poorly understood. In a petal loss (ptl) mutant, the
orientation and growth of the second whorl organs are aberrant, even if the
identity of the second whorl organs is changed
(Griffith et al., 1999). Thus,
PTL might be one of the regulators involved in second whorl organ
development, which is independent of the organ identity. Some other factors
are involved in petal growth because petal primordia initiate and aberrant
petals form in ptl mutants.
We describe analyses of a novel mutant, rabbit ears (rbe), which has defects in petal development. A cloning and expression analysis of RBE reveal that it is a transcriptional factor responsible for the development of the second whorl organs independently of the organ identity.
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Materials and methods |
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Microscopy
For scanning electron microscopy, samples were prepared as previously
described (Matsumoto and Okada,
2001). For histological analysis, inflorescences were fixed in
FAA, dehydrated in ethanol and embedded in Paraplast. Serial, 8 µm sections
were deparaffinized and stained in 0.1% Toluidine Blue.
Mapping and cloning of the RBE gene
F2 plants, which were obtained by crossing rbe-1 and
Col, were used for mapping. The DNA markers used for positional cloning were
based on SSLP (simple sequence length polymorphism) and CAPS (cleaved
amplified polymorphic sequence) between ecotypes Ler and Col.
Information about nga225, PAI2, ASA1, 217C, n97067 and nga249 markers was
obtained from The Arabidopsis Information Resource
(http://www.arabidopsis/org/).
After RBE had been mapped between the PAI2 and 217C markers, we
isolated recombinants by using these two markers among about 4000
F2 plants. Subsequently, we identified the relationship between the
RBE locus and these markers by analyzing the phenotypes of each
recombinant. Based on the sequence data from the Kazusa Arabidopsis Data
Opening Site
(http://www/kazusa/or.jp/kaos)
and on the Ler genomic sequence, we found several polymorphisms
between Ler and Col, which were then synthesized as sequence markers.
The primer sequences were: m1, 5'-AGAGGTGGTTATGTCAGTGC-3' and
5'-GATACACATCAGGGCCAATC-3'; m2,
5'-CCACAAGGATTGACAGAAAC-3' and
5'-GGAGATATCTAGCCTCTTCC-3'; m3,
5'-TCGTAGCCTAGCGAAGAAAG-3' and
5'-GGCAGTTGCTGATATCAGTC-3' and m4,
5'-CTACTAGAGCTTGAGGATGC-3' and
5'-GATGCTGACGTTGATATCCC-3'. cDNA cloning was performed by both
5'-RACE and 3'-RACE using the SMART RACE cDNA Amplification Kit
(Clontech). Sequencing of the PCR-amplified fragment and subcloned inserts was
performed by ABI Prism BigDye Terminator Cycle Sequencing Ready Reaction Kits
and ABI Prism 310, 377 and 3100 from Applied Biosystems.
Complementation test
The genomic fragment including the open reading frame (ORF) of RBE
was amplified by PCR (primer sequences
5'-CCTTTAAAGGCTCTCTCGTCTCTCTGTATT-3' and
5'-CTCACATCTTCGCTCTTCATCAACAGGTCT-3'), digested by
EcoT22I and SalI, and subcloned into the pPZP211 binary
vector (Hajdukiewicz et al.,
1994) to generate pRGEN. The pRGEN was transformed to the
rbe-1 mutant by a vacuum infiltration procedure with the
Agrobacterium strain C58C1. Transgenic plants were screened on an
agar medium containing 30 µg/ml kanamycin and 100 µg/ml
carbenicillin.
Transient expression analysis of the RBE-GFP fusion protein
Full length RBE ORF without the stop codon was amplified by PCR
(primer sequences 5'-ATCTCTAGAATGATGGATAGAGGAGAATG-3' and
5'-TAAGGATCCACCTCCGTTAACCTTAGGCGGATCAGCTCC-3'), digested by
XbaI and BamHI and cloned into pBluescriptII SK+
(Stratagene) to generate pRFSK. The fragment of G3GFP
(Kawakami and Watanabe, 1997)
was amplified by PCR (primer sequences
5'-AGTGGATCCGGTGGAAGTAAAGGAGAAGAAGAACTTTTC-3' and
5'-CCACCGCGGTTATTTGTATAGTTCATGCATGCC-3'), digested by
BamHI and SacII and cloned into pBluescriptII SK+ to
generate pG3SK. The BamHI-SacII fragment from pG3SK was
subcloned into pRFSK to generate pRFG3SK. The XbaI-SacII
fragment from pRFG3SK containing the RBE-G3GFP fusion gene was
subcloned into pGEM-3Zf+/35S-NosT to generate pRFG3GFP. The
HindIII-EcoRI fragment from pRFG3GFP, which contains the
cauliflower mosaic virus (CaMV) 35S promoter, the RBE-G3GFP
fusion gene and the nopaline synthase (NOS) terminator, was
subcloned into pBI121 (Clontech) to generate pRFG3BI. pRFG3BI was transformed
into an Arabidopsis Col-0 cell suspension and screened as previously
described (Mathur et al.,
1998
). Samples were visualized under an Axiophoto2 microscope
(Zeiss) with a FITC filter, and photographed with a Nikon COOLPIX 990 digital
camera.
RNA isolation and RT-PCR
Total RNA was isolated with the Isogen reagent (Nippon gene). One microgram
of total RNA from each tissue was reverse-transcribed using the SUPERSCRIPTII
reverse transcription kit (Invitrogen), and 1.0 µl of cDNA was used as a
template for PCR. The products were electrophoresed on an agarose gel,
transferred to nylon membranes and hybridized with RBE and
ACT8 probes. A part of the RBE cDNA (corresponding to the
sequence at 152-400) was labeled and used for the RBE probe. Specific
primers for the detection of ACT8 mRNA were generated as described
previously (Aida et al.,
1997).
mRNA in situ hybridization
mRNA in situ hybridization was performed as previously described
(Matsumoto and Okada, 2001). A
part of RBE cDNA (corresponding to the sequence at 272-579) was
amplified by PCR and cloned into pBluescriptII SK+ at the EcoRV site
for the antisense and sense probes. For the probe of the APETALA3
(AP3), a part of AP3 cDNA (corresponding to the sequence at
535-820, including the 3' UTR region) was amplified by PCR, and cloned
into pBluescriptII SK+ at the EcoRV site.
Promoter analyses of the RBE gene
The XbaI-EcoRI fragment from pERGSK, which contained
G3GFP with ER target signal sequences, was ligated into pBI101 (Clontech) to
generate pERGBI. The RBE promoter was amplified by PCR (primer
sequences 5'-AAACCGCGGTTTCAAGCAGTCTGATCACG-3' and
5'-TTTTCTAGACAGTAGAAGAAGTTAA-GGTG-3'), digested by SacII
and XbaI, and then cloned into pBluescriptII SK+ to generate pRPSK.
The HindIII-XbaI fragment from pRPSK was then subcloned into
pERGBI and pBI101 to generate pRPERGBI and pRPGUS, respectively. To construct
the RBE promoter::diphthria toxin (RBEp::DT-A), DT-A was
amplified by PCR from pRDC4, digested by XbaI and SacI, and
ligated into pBluescriptII SK+ to generate pDTASK. The
XbaI-SacI fragment of pDTASK was then subcloned into pRPGUS
to generate pRPDTABI. Transformation to plants and screening were performed as
described above. GUS staining was performed as described previously
(Donnelly et al., 1999).
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Results |
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A histological analysis was performed to investigate the early defects in
rbe. In wild-type plants, L2 or L3 cells of petal precursor cells
divide periclinally (Hill and Lord,
1989) and petal primordia bulges emerge up to stage 6
[Fig. 1K; flower stages as
defined by Smyth et al. (Smyth et al.,
1990
)]. In rbe-2, cell division of the petal precursor
cells was arrested and no primordia formed where the petal primordia should
have emerged (Fig. 1L). In a
wild-type stage 10 flower, petals elongate and reach the height of anthers
(Fig. 1M). At the same stage of
an rbe-2 flower, no petals formed because of the loss of petal
primordia (Fig. 1N). This
suggests that the initiation of petal primordia does not occur in rbe
and that RBE is involved in the early development of petal
primordia.
We noticed that, in many cases in rbe-1, two adjacent petals remained normal, but the other two petals were deformed (Fig. 1B,C; the mutant name is derived from this phenotype). To determine whether this phenotype is position dependent, we scored the phenotype of the four petals based on their positions relative to the inflorescence meristem (Fig. 1J, Table 1). Interestingly, in rbe-1, flowers formed immediately after bolting were almost normal (data not shown), but aborted petals became prominent in flowers formed later. When we examined the petal shape of the sixteenth to twentieth flowers on the inflorescence of rbe mutants, the two petals on the adaxial side (position 3 and 4) were deformed more severely than those on the abaxial side (position 1 and 2) (Table 1). Indeed, in rbe-1 about 60% of the petals at position 1 and 2 remained normal, whereas only about 30% of the petals at position 3 and 4 were normal. These difference between the adaxial and the abaxial sides was also found in rbe-2 (Table 1).
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The outer and inner integuments of rbe were shorter than those of the wild-type, causing a reduction of mature seed number and the length of siliques (data not shown). In the other floral organs and vegetative tissues, rbe was indistinguishable from the wild type.
RBE encodes a SUP-like zinc finger protein
We found mutations in three ORFs residing in the region where RBE
was mapped on chromosome 5 (Fig.
2A). 2.5 kb of genomic DNA including one of the ORFs, At5g06070,
complemented the rbe phenotype
(Fig. 2A and data not shown),
indicating that this ORF is the RBE gene. In rbe-1, a C to T
transversion at the nucleotide position 232 in the cDNA sequence results in
the replacement of an arginine with a stop codon at amino acid 78 in the RBE
protein. In rbe-2, which was isolated from the SIGnAL T-DNA insertion
stocks (accession number is Salk037010), a T-DNA was inserted into nucleotide
position 428, causing a deletion of 19 nucleotides
(Fig. 2B).
|
The RBE protein is located in the nucleus
Zinc-finger proteins are suggested to function as transcriptional factors
(Takatsuji, 1998), and the RBE
protein contains the potential nuclear localization signal (RRDRAR) just after
the zinc finger domain (Fig.
2B) (Dinkins et al.,
2003
). To confirm that RBE is located in the nucleus, an RBE-GFP
fusion protein was expressed transiently in suspension culture cells of
Arabidopsis under the CaMV 35S promoter. As shown in
Fig. 3, the RBE-GFP fusion
protein was located in the nucleus. The same results were obtained when the
same construct was introduced to onion epidermal cells (data not shown). These
results suggest that RBE is a putative transcriptional factor.
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In stage 12 flowers of the wild type, petal length reached the anther of longer stamens (Fig. 6A,B). In contrast, plants carrying the RBEp::DT-A transgene lacked petals completely in flowers of the same stage (Fig. 6C,E), and traces of petal primordia were not observed in mature flowers (Fig. 6D,F). These results indicate that RBE is expressed in cells that could be involved in, or recruited to, the petal primordia. The other floral organs were not affected, indicating that the ablation of the cells expressing RBE did not affect the development of the other floral organs in both the outer and inner whorls.
|
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To investigate whether RBE is expressed in the petal primordia
formed at abnormal positions, RBE expression was analyzed in the
flowers of mutant or transgenic plants that had ectopic petals. In flowers of
35S::PI 35S::AP3 double transgenic plants that have petals in the
first whorl instead of sepals (Krizek and
Meyerowitz, 1996), RBE expression was not observed in the
petal primordia in the first whorl (shown as p' in
Fig. 7K), although normal
expression was observed in the petal primordia of the second whorl
(Fig. 7L). The flowers of
agamous-1 (ag-1) mutants are known to have homeotic
conversion of six stamens to petals in the third whorl, and have a meristem of
indeterminate character that continuously forms a flower of the three whorls
in place of the fourth whorl (Bowman et
al., 1989
; Bowman et al.,
1991
). When the expression of RBE was examined in an
ag-1 flower, the signal was detected in the cells corresponding to
the petal primordia in the fifth whorl
(Fig. 7N) in addition to those
in the second whorl (Fig. 7M), but not in the primordia of homeotically converted petals in the third whorl
(Fig. 7M). These results are
consistent with the previous observation that RBE expression is
restricted to the organ primordia in the second whorl independently of the
organ identity.
RBE is not expressed in ap1-1 and ptl-1
To investigate whether RBE is expressed in flowers that lack
petals, in situ hybridization was performed in the continuous sections of
inflorescences of petal-defective mutants, apetala1-1
(ap1-1) and petal loss (ptl). ap1-1 has
leaf-like organs in place of the sepals, usually no petals and reduced number
of stamens (Irish and Sussex,
1990). Although ap1-1 mutant flowers looked to have a
region corresponding to the second whorl, RBE was not expressed
(Fig. 7O). In flowers of
another petal-defective mutant, ptl, RBE was not expressed in the
region corresponding to the primordia of the second whorl
(Fig. 7P). This suggests that
RBE expression is under control of AP1 and PTL.
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Discussion |
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RBE could function either by promoting precursor cells to form petal primordia or by recruiting cells of other regions to make petal primordia. To investigate these possibilities, we generated transgenic plants carrying APETALA3 promoter::RBE (AP3p::RBE). In the transgenic plants, organ primordia did not increase in the second and third whorls, and the flowers were indistinguishable from those of the wild type (data not shown). We then generated 35S::RBE plants that produced flowers in which a change of floral organ number and arrangement was not observed (data not shown). These results indicate that petal precursor cells are susceptible to RBE action, but the other cells in the second whorl and other whorls are not.
How are the positions of the primordia determined in floral whorls? RBE was not expressed in the ap1-1 mutant, indicating that RBE acts downstream of AP1. However, AP1 does not seem to regulate the position of primordia, because it is expressed in the entire first and second whorls. Furthermore, the mutation of RBE did not affect the arrangement of the primordia, and RBE was expressed in four-petal-precursor cells, which suggests that RBE is not involved in determining the position of petal primordia. We present a model in which an unknown gene, `X', acts downstream of AP1, and is involved in positional determination and regulation of the expression of RBE in the petal precursor cells. RBE was not expressed in ptl-1, implying that PTL could be the unknown X gene. Gain-of-function analyses of PTL would shed light on the positional determination of petal primordia in a flower.
Abnormal petals in both rbe-1 and rbe-2 formed more frequently on the adaxial than abaxial side of the flower (Table 1). This tendency was also observed in ptl-1 (Table 1), suggesting that petal development is regulated differently on the adaxial and abaxial sides in Arabidopsis. It is unlikely, however, that RBE and PTL promote the development of petals on the adaxial side more than petals on the abaxial sides because (1) RBE transcripts were expressed at the same time and intensity in all four petal primordia and their precursor cells, and (2) the abnormality was incomplete in these mutants; that is, normal petals were sometimes formed even on the adaxial side and abnormal petals were frequently formed on the abaxial side (Table 1). We supposed that the growth rate of petals on the two sides is controlled differently in Arabidopsis, and that RBE and PTL have a role in adjusting the rate to keep them even.
It has been shown that the first and fourth whorl organs are not affected
when the second and third whorl organs are ablated genetically
(Day et al., 1995). We have
shown that the genetic ablation of the second whorl organs does not affect the
development of other floral organs (Fig.
6). This is consistent with the prediction that some mechanism
that defines the position of organ primordia in all four whorls and the timing
of the primordia development is settled before stage 3, when the expression of
RBE and AP3 starts (Fig.
5C) (Day et al.,
1995
; Hicks and Sussex,
1971
). Further analyses, such as genetic ablation of organ
primordia in other whorls, would be required to confirm this hypothesis.
The phenotype of the rbe-1 ap3-5 double mutants was additive
(Fig. 7C,D), which indicates
that RBE functions independently of organ identity determination. Our
in situ analyses also showed that determination of position and identity of
floral organs is regulated by independent mechanisms
(Fig. 7E-N). In the
ap3 and pi mutants, even though the second whorl petals are
transformed into sepalloid organs, the size of their primordia is almost the
same as that of petals, but not as large as sepal primordia observed in
wild-type floral buds (Bowman et al.,
1989; Hill and Lord,
1989
). This indicates that floral homeotic genes determine organ
identity, but they are not involved in the determination of the size of floral
primordia or in their growth. RBE and PTL might be involved
in, or regulated by, this pathway.
We also constructed rbe-1 clv1-4 and rbe-1 pan double mutants. In these double mutants, the phenotype of the organs formed in the second whorl was largely additive. The organs were small and under-developed, as shown in rbe-1, although their number changed as expected from the second mutation of each double mutant (data not shown). This suggests that the role of RBE in petal development is independent of the genes that control organ number.
Molecular function of one-finger type zinc finger proteins
RBE encodes a nuclear-localized one-finger type Cys2His2 zinc
finger protein (Figs 2,
3). According to the amino acid
sequence of the zinc finger motif, RBE belongs to the EPF family of proteins,
which contains the QALGGH amino acid residues within the zinc finger motif
(Takatsuji, 1998;
Takatsuji, 1999
). In this EPF
family, ZPT2-1 (or EPF1) of Petunia with two
Cys2His2 zinc finger motifs has been well analyzed. It interacts with the
promoter region of 5-enolpyruvylshikimate-3-phosphate synthase (EPSPS) and is
involved in the activation of transcription
(Takatsuji et al., 1992
). The
DNA binding site of two finger proteins is specified by the linker sequence
length between the two zinc finger domains
(Takatsuji and Matsumoto,
1996
). Recently, it has been shown that SUP can bind EPF-binding
target sequences, and that amino acid residues around the zinc finger motif
are involved in the recognition of the target sequence
(Dathan et al., 2002
). These
data indicate that both one- and two-finger proteins bind DNA, although their
mechanisms of binding site recognition are different. The amino acid residues
around the zinc finger motif show a low similarity between RBE and SUP (data
not shown). Thus, RBE might bind DNA as well as SUP, but the residues around
the zinc finger motif would be involved in the recognition of different
binding-target DNA sequences.
Expression patterns of RBE and SUP in a floral bud are
exclusive, suggesting that their functions are divided spatially. In addition,
the function of these two genes is likely to be antagonistic in respect to
floral development; SUP has been suggested to regulate cell
proliferation negatively at the boundary of the third and fourth whorls
(Sakai et al., 1995;
Sakai et al., 2000
), and outer
integuments of sup go through excess elongation
(Gaiser et al., 1995
).
However, RBE was expressed in petal primordia and their precursor
cells (Fig. 5), and its
mutation results in arrested cell division of petal precursor cells and in
deformed petals (Fig. 1). In
addition, integuments of rbe were shorter than those of the wild type
(data not shown). These results suggest that RBE is involved in cell
specification or the activation of cell proliferation, rather than in
repression of cell proliferation, like SUP. Thus, in the flower, not
only the functional region, but also the target genes of RBE and
SUP would be different. We found that another gene in the
RBE-SUP family was expressed in inflorescences with young floral buds
and mature flowers (data not shown), presenting the possibility that this
additional gene is also involved in floral organ development. Analysis of this
gene would help to understand the function of the single zinc finger genes
belonging to the RBE-SUP family.
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ACKNOWLEDGMENTS |
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Footnotes |
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