Plant Molecular Biology 56: 133–143, 2004.
2004 Kluwer Academic Publishers. Printed in the Netherlands.
133
Ectopic expression of OsYAB1 causes extra stamens and carpels
in rice
Seonghoe Jang1,3, Junghe Hur1, Soo-Jin Kim2, Min-Jung Han1, Seong-Ryong Kim2
and Gynheung An1,*
1
National Laboratory of Plant Functional Genomics, Division of Molecular and Life Sciences, Pohang University of Science and Technology, Pohang 790-784, Republic of Korea (*author for correspondence; e-mail
[email protected]); 2Department of Life Science, Sogang University, Seoul 121-742, Republic of Korea;
3
Present address: Department of Developmental Biology, Max-Planck-Institute for Plant Breeding Research,
Carl-von-Linne-Weg 10, 50829 Cologne, Germany
Received 21 January 2004; accepted in revised form 30 August 2004
Key words: ectopic expression, flower development, nuclear protein, rice, YABBY family
Abstract
Members in the YABBY gene family of proteins are plant-specific transcription factors that play critical
roles in determining organ polarity. We have isolated a cDNA clone from rice that encodes a YABBY
protein. This protein, OsYAB1, is similar to Arabidopsis YAB2 (50.3%) and YAB5 (47.6%). It carries a
zinc-finger motif and a YABBY domain, as do those in Arabidopsis. A fusion protein between OsYAB1 and
GFP is located in the nucleus. RNA gel-blot analysis showed that the OsYAB1 gene is preferentially
expressed in flowers. In-situ hybridization experiments also indicated that the transcript accumulated in the
stamen and carpel primordia. Unlike the Arabidopsis YABBY genes, however, the OsYAB1 gene does not
show polar expression pattern in the tissues of floral organs. Our transgenic plants that ectopically expressed OsYAB1 were normal during the vegetative growth period, but then showed abnormalities in their
floral structures. Spikelets contained supernumerary stamens and carpels compared with those of the wild
types. These results suggest that OsYAB1 plays a major role in meristem development and maintenance of
stamens and carpels, rather than in determining polarity.
Introduction
Proteins in the YABBY family are transcription
factors that contain a zinc-finger domain in the
amino-terminal region and a YABBY domain in
the carboxyl-terminal region. The latter is similar
to the first two helices of the HMG box. That box
is a DNA binding motif found in a high-mobility
group of non-histone chromosomal proteins and
other vertebrate transcription factors, such as
SRY, and is likely to form a helix-loop-helix
structure (Bowman and Smyth, 1999). The
YABBY family transcription factors appear to be
plant-specific; genes with a similar juxtaposition of
zinc finger and YABBY domain do not occur
in other kingdoms (Golz and Hudson, 1999;
Bowman, 2000).
At least six YABBY genes have been identified
in the Arabidopsis genome (Bowman and Smyth,
1999). For example, in INNER NO OUT (INO;
YAB4) mutant plants, the outer integument fails to
differentiate on the abaxial side of the ovule and,
subsequently, does not develop. Although a single
mutation in CRABS CLAW (CRC, Bowman,
2000), YABBY 3 (YAB3), or FILAMENTOUS
FLOWER (FIL; YAB1) produces no loss of polar
differentiation, their double-mutant combinations
demonstrate that they are also responsible for
abaxial cell identity. In FIL and YAB3 double
mutants, the cotyledons and leaves are more linear
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than the normal wild-type ovate form, and are
occassionally bifurcate. The abaxial leaf surfaces
resemble a mosaic of abaxial and adaxial tissues.
In double-mutant flowers, nearly all floral organs
are somewhat radicalized (Siegfried et al., 1999;
Kumaran et al., 2002). Moreover, the crc and
gymnos/pickle or crc and kanadi double mutants
show ectopic development of adaxial tissues in
abaxial positions (Eshed et al., 1999). These
observations indicate that the juxtaposition of
abaxial and adaxial cell fates is required for lamina
outgrowth.
Most transgenic plants that ectopically express
the FIL gene die after forming a few rosette
leaves; abaxialization of tissues is present in their
cotyledons and rosette leaves (Sawa et al.,
1999b). Similarly, over-expression of YAB3 and
CRC causes ectopic abaxial tissues to develop in
lateral organs, e.g., the cotyledons and leaves
(Alvarez and Smyth, 1999; Siegfried et al., 1999).
Siegfried et al. (1999) have observed that the
formation of shoot apical meristems (SAM) ceases or is arrested in transgenic plants overexpressing either FIL or YAB3, which may be a
result of severe abaxialization. In transgenic
flowers that ectopically express CRC, ovules and
stigmata develop along the sepal margins; carpels
that arise as small, solid cylindrical structures are
composed primarily of style tissue topped with a
stigma. Their floral meristems also fail to produce
the full complement of organs while their apical
inflorescence meristems often terminate in a carpelloid structure (Alvarez and Smith, 1999).
Therefore, these observations suggest that YABBY family genes act primarily in cellular
polarity.
Transcripts of FIL have been detected from the
sub-epidermal cells of the presumptive cotyledons,
leaves, floral meristems, or floral organ initials
within the apical meristems; they are restricted to
the abaxial regions of organ primordia as they
emerge from the meristems, and are maintained
even in mature floral organs, such as the sepal,
petal, stamen, and carpel. YAB2 and YAB3 transcripts are qualitatively expressed in a similar
manner to FIL, but they do differ quantitatively,
i.e., FIL is strongly expressed; YAB3, moderately;
and YAB2, weakly (Siegfried et al., 1999). Other
Arabidopsis YABBY genes, such as CRC and INO,
show similar polar expression, although CRC
mRNAs are limited to the nectaries and carpels
while INO is found only in the outer integuments
(Bowman and Smyth, 1999; Siegfried et al., 1999;
Villanueva et al., 1999).
The rice flower has an architecture different
from that of the model dicot species. It does not
have petals. Instead, two bag-shaped lodicules are
located at one side of the inner wholes containing
carpels and stamens, and they are subtended by
palea and lemma, bract-like structures that enclose
the floral organs. Therefore, it will be interesting to
investigate functional roles of YABBY genes in
rice. Recently, Yamaguchi et al. (2004) identified
the responsible gene for drooping leaf (dl ) mutation in rice. The DL gene encodes a YABBY
protein, which has an essential role in specifying
carpel identity and meristem determinacy in rice
flowers and also regulating midrib formation in
leaves. In this study, we examine the role of another rice YABBY gene, OsYAB1 in the formation
of floral organs.
Materials and methods
Bacterial strains and plant material
Escherichia coli JM109 served as the recipient for
routine cloning experiments on japonica rice
cultivar ‘Dongjin’. We used Agrobacterium tumefaciens LBA4404 containing the Ach5 chromosomal background and a disarmed helper-Ti
plasmid pAL4404 (Hoekema et al., 1983). All
transformation procedures were performed as
described previously (Lee et al., 1999; Jeon et al.,
2000).
Molecular cloning
A cDNA clone containing the full open reading
frame (ORF) of OsYAB1 was generated by PCR.
The forward primer sequence was 50 -CGGTCTAGAAATGTCGGTCCAGTTTAC-30 , which contains
the ATG start codon. The reverse primer sequence was
50 -CGCCTCGAGTGTCTACGTACATAGCACAGC-30 , located 263 bp downstream from the stop codon.
Additionally, a cDNA fragment comprising part of the
ORF and the untranslated region was amplified for
antisense construction using Primers 50 -GCGCTCGAGC-AAGGAGGAGATACAGAG-30 and 50 - CCGTCTAGATGTCTACGTACATAGCACAGC-30 . For
subsequent cloning, each primer contained an
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XbaI or XhoI site in its 50 end. The PCR fragment
was inserted into the pGEM-Teasy vector
(Promega, Madison, WI) and the clones were sequenced to check for possible amplification errors.
Binary vector pGA1671 (Jeon et al., 2000; Kim
et al., 2003) was used for assembling the sense and
antisense constructs. This vector possesses the
hygromycin phosphotransferase (hpt) gene as a
selectable marker under the control of the cauliflower mosaic virus 35S promoter followed by the
termination region of the 7 gene of pTiA6. It also
has three unique sites (XbaI, MluI, and XhoI)
between the rice actin1 promoter region, containing the first intron (McElroy et al., 1990), and the
nopaline synthase terminator (An et al., 1988).
Thus, this vector can be used for expressing a
foreign gene in monocot plants when transferred
by the Agrobacterium co-cultivation method. The
OsYAB1 full ORF and the partial cDNA were
inserted between the XbaI and XhoI sites in the
sense and antisense orientations, thereby constructing pGA2786 and pGA2787, respectively.
To obtain a fusion gene between OsYAB1 and
GFP, we replaced the stop codon of OsYAB1 with
a BamHI restriction site via PCR, using Primers
50 -GCGTCTAGAGAGAAGATGGGGAGGG
GG-AAGATC-30 and 50 -GCCGGATCCAAAC
ACC-AAAAATAATTGAAGGCCGGC-30 . After the
amplified fragment was inserted between XbaI and
BamHI of the pCaMV35S-GFP vector (Jung
et al., 2002), an in-frame fusion was generated
between the OsYAB1 and GFP genes. The clone
was then sequenced to check for possible amplification errors.
RNA expression analyses
Total RNAs were isolated from various vegetative
organs and panicles at different developmental
stages, using an RNA isolation kit (Tri Reagent;
MRC Inc., Cincinnati, OH). The isolated total
RNAs were fractionated on a 1.3% agarose gel,
blotted onto a nylon membrane (Hybond N+;
Amersham, Buckinghamshire, UK), and hybridized with a 32P-labeled probe.
For semi-quantitative RT-PCR analyses
(Leblanc et al., 1999), 10 lg of total RNA was
reverse transcribed in a total volume of 50 ll that
contained 1 lg of oligo(dT)15 primer, 2.5 mM
dNTPs, and 200 units of Moloney murine leukemia virus Reverse Transcriptase (New England
Biolabs, Beverly, MA) in a reaction buffer. PCR
was performed in a 50 ll solution containing a 1 ll
aliquot of the cDNA reaction, 0.2 lM gene-specific primers, 10 mM dNTPs, 1 unit of rTaq DNA
polymerase (TakaRa Shuzo, Shiga, Japan), and 10
· reaction buffer. PCR conditions for each cycle
included 0.5 min at 94 C, 0.5 min at 57 C, and
0.75 min at 72 C. For each cDNA, 20 to 25 cycles
were performed. RT-PCR primers for OsYAB1
are OsYAB1-f (50 - CTATTGCAACACTATC
CTT-GTG- 30 ) and OsYAB1-r (50 - GACGT
ATAGGT-GACACTTGCTG-30 ). The amplified
266-bp fragment at the 50 end of the OsYAB1
cDNA, excluding the YABBY domain, served as a
probe for the RNA gel-blot analysis and in situ
hybridization experiments. Rice actin1 cDNA was
used for the normalization of RT-PCR (McElroy
et al., 1990; Jeong et al., 2003). Here, 10 ll of the
reaction mixture was separated on a 1.6% (w/v)
agarose gel and transferred to a nylon membrane.
All procedures for these blot analyses were previously described by Kang et al. (1997).
In-situ localization of the transcript
Spikelets were fixed overnight at 4 C in 2% (W/V)
paraformaldehyde plus 2.5% (V/V) glutaraldehyde in 50 mM PIPES buffer (pH 7.2). The fixed
tissues were dehydrated by a graded concentration
of ethanol and embedded in a Paraplast Tissue
Embedding Medium (Paraplast X-tra; Oxford
Labware, St. Louis, MO). These tissues were then
sliced into 7-lm sections with a rotary microtome
(Leica, Bannockburn, IL). The sections were attached to silanized glass slides (Matsunami Glass,
Tokyo, Japan). Afterward, the paraffin was removed through a graded ethanol series, and the
samples were dried for 1 h. Digoxygenin-labeled
sense or antisense RNA probes were prepared
from the linearized pBluscript carrying partial
OsYAB1 cDNA, using either T3 or T7 RNA
polymerase. The sections were hybridized with the
probes at 48 C for 16 h in a hybridization solution, then washed in solutions containing 2.0 ·
SSC, 1.0 · SSC, and 0.1 SSC, for 15 min each, at
50 C. The hybridizing probes were detected colorimetrically with an anti-DIG conjugated alkaline phosphatase (Roche Molecular Biochemicals,
Mannheim, Germany). Photographs were taken
under a bright-field microscope (Nikon Eclipse
600, Melville, NY).
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Light Microscopy
Spikelets were prepared in a fixative solution of
50% ethanol, 0.9 M glacial acetic acid, and 3.7%
formaldehyde for 15 h at 4 C. They were then
dehydrated with ethanol, infiltrated with xylene,
and embedded in paraffin (Paraplast X-tra).
Afterward, 12-lm-thick sections were transferred
onto gelatin-coated glass slides, deparaffinized in
xylene, and rehydrated in a graded ethanol and
water series. These sections were stained in 0.1%
toluidine blue O (Sigma, St. Louis, MO, USA),
dehydrated with ethanol, infiltrated with xylene,
and covered permanently. Light microscopy was
performed with a Nikon Labophoto-2 (Nikon,
Tokyo, Japan).
GFP imaging
The plasmid DNAs were purified on a Qiagen
column (Qiagen, Valencia, CA) according to the
manufacturer’s instructions. Plasmid DNAs from
the fusion constructs were precipitated onto
1.5 mg of 1-lm gold particles (BioRad, Hercules,
CA). The particles were then re-suspended in ethanol and divided into three aliquots. Slices of
onion scale were bombarded using the PDS-1000
System (BioRad), at 1100 psi helium pressure.
Expression of the fusion constructs was monitored
at 24 to 36 h after bombardment via fluorescence
microscopy, using an Axioplan fluorescence
microscope (Zeiss, Jena, Germany). Images were
captured with a cooled charge-coupled device
camera. The data were processed with Adobe
Photoshop software (Mountain View, CA) and
presented in pseudocolor format.
Results
Cloning OsYAB1 cDNA
We amplified a cDNA clone carrying the fulllength ORF, using primers designed based on the
partial cDNA clone, AF098752. The amplified
PCR fragment was inserted into the pGEM-Teasy
cloning vector and the nucleotide sequence was
deduced. Our cDNA clone is 920 bp long and
contains an ORF of 169 amino acid residues
(Figure 1A). We have designated this rice clone as
OsYAB1. The OsYAB1 protein shows sequence
identity to YAB2 (50.3%), OsYAB2 (49.7%),
YAB5 (47.6%), CRC (40.3%), FIL (39.1%),
YAB3 (37.0%) and DL (36.3%). The OsYAB1
protein carries the C2C2 -type zinc finger motif at
the residues between 11 and 46. It also carries the
YABBY domain, which forms a potential helixloop-helix structure. In the public databases, six
rice YABBY genes are present: OsYAB1 (accession no. AAC72847), OsYAB2 (AAS07125), OsYAB3 (NP_922256), OsYAB4 (NP_911282),
OsYAB5 (CAD41530), and DL (BAD06552).
Comparison with the proteins in the YABBY
family indicated that all of these proteins except
OsYAB4 share the zinc finger motif and the
YABBY domain. An unrooted dendrogram using
neighbor-joining method (Saitou and Nei, 1987;
www.genome.ad.jp) revealed close relations
among the proteins. OsYAB1 is clustered with
OsYAB2, YAB2, and YAB5 (Figure 1B). Previously, Goff et al. (2002) reported five YABBY
genes in rice. OsYAB1 comprises six exons and is
located at 24.8 cM on Chromosome 7 (http://
rgp.dna.affrc.go.jp).
Expression analyses
Total RNAs were extracted from various vegetative organs and panicles at different developmental
stages. Transcript levels of the OsYAB1 gene were
determined via RNA-gel blot analysis. Here, we
used a probe prepared from the 266-bp PCR
fragment containing a partial zinc finger motif but
no YABBY domain. The 1.2-kb OsYAB1 transcript was most strongly detected in immature
panicles and was also weakly found in mature
panicles (Figure 2A). Semi-quantitative RT-PCR
analyses also showed that the OsYAB1 gene was
expressed throughout panicle development (Figure 2B), with its expression level being strong in
the immature panicles and decreasing as fruits
developed. This gene was also expressed at low
levels in vegetative organs, e.g., two-week-old
seedling shoots and flag leaves.
Because OsYAB1 is preferentially expressed in
reproductive organs, we compared its pattern with
that of the MADS box genes, which play a major
role in controlling reproductive development
(Figure 2B). The OsYAB1 expression pattern was
most similar to that of OsMADS15 and OsMADS3. OsMADS15, an AP1 (APETALA 1)
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Figure 1. A. Alignment of OsYAB1 and related protein sequences. The zinc finger domain (shaded box), the putative component of
the zinc finger motif (dark circles) and the HMG-box domain (underlined bold letters) are indicated. Identity to OsYAB1 is shown at
the end of the sequences in parentheses. Dashes were introduced for maximum sequence homology. Asterisks represent amino acid
residues identical to corresponding ones in OsYAB1. Numbers on right represent positions of amino acid residues shown for each
sequence. B. Unrooted dendrogram for OsYAB1 and related proteins, as generated by neighbor-joining method (Saitou and Nei,
1987). Proteins are as follows (accession numbers in parentheses): OsYAB1 (AF098753), OsYAB2 (AAS07125), FIL (AF136538),
YAB2 (AF136539), YAB3 (AF136540), YAB5 (At2g26580), INO (AF195047), CRC (AF132606), OsYAB3 (NP_922256), OsYAB4
(NP_911282), OsYAB5 (CAD41530), DL (BAD06552), TaYAB (wheat YABBY protein, AAQ93323), ZYB14 (maize YABBY protein, AAP79884), ZYB10 (maize YABBY protein, AAP79887).
homolog, is essential for floral organ initiation
and maintenance (Kyozuka et al., 2000). The
OsMADS3 gene is a C function homeotic gene
that controls stamen and carpel identity and
maintenance (Kang et al., 1998; Kyozuka and
Shimamoto, 2002).
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Figure 1B. (Continued).
Differences in OsYAB1 transcript levels among
various floral organ-defective mutants, such as
frizzy panicle, multiple pistil, and the OsMADS3
knock-out, as well as OsFOR1 antisense plants
were investigated (Librojo and Khush, 1985;
Komatsu et al., 2001; Jang et al., 2003; Lee et al.,
2003). However, we found no significant change in
transcript levels among the mutants, indicating
that OsYAB1 is located upstream of the genes in
the floral developmental sequence (data not
shown).
We also performed RNA in-situ hybridization
experiments to investigate the spatial expression
pattern of OsYAB1gene (Figure 3). In young
spikelets, OsYAB1 transcript was abundant in the
stamen and carpel primordia. Interestingly, unlike
with the Arabidopsis YABBY genes, the transcript
was not distributed in a polar manner.
Figure 2. A. Spatial expression pattern of OsYAB1 by RNA
gel-blot analysis. Upper: 15 lg of total RNAs isolated from
various organs was hybridized with the OsYAB1 gene-specific
probe. Lower: ethydium-bromide staining of rRNA, showing
the amount of sample used for analysis. B. Semi-quantitative
RT-PCR analyses of OsYAB1 and rice MADS-box genes.
OsMADS14, OsMADS15, and OsMADS18 are A-class
MADS-box genes (Moon et al., 1999) and OsMADS3 is a Cclass MADS-box gene (Kang et al., 1998). Rice actin1
transcript was amplified as a control. Primers for actin1 were
designed to amplify for exons flanking the 2nd intron; only the
231-bp cDNA fragment was amplified, demonstrating no
genomic DNA contamination in templates.
Ectopic expression of OsYAB1
The functional roles of OsYAB1 were studied by
ectopically expressing the gene in rice. Ectopic
expression system is one of the useful methods to
analyze the influence of expression of genes, which
regulate developmental processes. It has been
shown previously that ectopic expression of the
floral homeotic gene alters floral organ identity in
homologous (Prasad et al., 2001; Kyozuka and
Shimamoto, 2002) and heterologous system (Kang
et al., 1995; Nandi et al., 2000; Tzeng et al., 2002).
OsYAB1 cDNA was inserted between the rice actin (act1) promoter (McElroy et al., 1990) and the
Figure 3. in situ localization of OsYAB1 transcripts in wild type
young flower. Antisense (A) and sense (B) probes were
hybridized. Symbols: ca, carpel; l, lemma; p, palea; st, stamen.
Bars¼20 lm.
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transcript 7 terminator, and the chimeric molecule
was introduced via Agrobacterium-mediated
transformation. We obtained 100 independently
transformed plants. RNA-gel blot analysis (using
the OsYAB1 probe) with RNAs from mature
leaves showed that most of these plants ectopically
over-expressed the transgene (Figure 4A). Although they manifested normal phenotypes during
their vegetative growth period, additional stamens
and carpels were found in their spikelets. In contrast, other floral organ types, such as their palea/
lemma, lodicules, and glumes, were indistinguishable from those of the wild-type controls. In the
latter plants, flowers had two white lodicules at the
adaxial (lemma) side in Whorl 2, six stamens in
Whorl 3, and one gynoecium with two stigmas in
Whorl 4 (Figure 4B, D; Figure 5A, C). The
transgenic spikelets, however, had one or two extra stamens in the third whorl, and one to seven
additional carpels in the fourth whorl (Figure 4C,
E-G; Figure 5B, D, E). The percentage of alterations was correlated with the ectopic expression level of the introduced OsYAB1 cDNA
(Figure 4A; Table 1).
We also made more than 100 independent
transgenic plants expressing the OsYAB1 cDNA in
the antisense orientation. However, no phenotypic
alterations were observed in either their vegetative
or reproductive organs (data not shown).
Sub-cellular localization of OsYAB1
Because YABBY proteins are considered
transcription factors, one would expect to find
them in the nucleus. Therefore, to examine whether OsYAB1 is a nuclear protein, we linked the
plant-optimized GFP gene to the C terminus of
OsYAB1 by modifying the stop codon to the
BamHI site where the GFP coding region was inserted. This OsYAB1–GFP fusion construct was
placed under the 35S promoter and introduced
Figure 4. Phenotypes of wild-type flowers and transgenic
flowers over-expressing OsYAB1. A. 15 lg of total RNA was
isolated from mature leaves of wild-type (WT) or transgenic
plants constitutively expressing OsYAB1 (S1–S5), and hybridized with the OsYAB1 gene-specific probe. Wild-type flowers
(B, D) consist of a carpel, two lodicules, six stamens surrounded
with palea/lemma, and a pair of glumes. Palea alone (B) or
palea/lemma (E) was removed for observation of inner organs.
Additional stamens (E) and carpels (C, F, and G) in transgenic
flowers are marked with arrows. Symbols: an, anther; ca, carpel; g, glume; lo, lodicule; p, palea. Bars¼1 mm.
Figure 5. Cross-sections of wild-type flowers and transgenic
flowers over-expressing OsYAB1. Wild-type flowers with either
six stamens or an ovary with a single ovule are shown in A and
C, respectively. Additional stamen is indicated with an arrow in
B, and double or triple carpels are marked with arrows in D and
E. Arrowhead in E represents separating point for independent
carpels. Symbols: an, anther; f, filament; l, lemma; o, ovary; ov,
ovule. Bars¼500 lm (A, B), 100 lm (C–E).
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Table 1. Floral organ numbers in wild-type plants and
transgenic plants over-expressing OsYAB1.
a
palea/
Plants No. of
lemma
flowers
examineda
lodicule
stamen
carpel
WT
S1
S2
S3
S4
S5
2.0±0.0
2.0±0.0
2.0±0.0
2.0±0.0
2.0±0.0
2.0±0.0
6.0±0.0
6.2±0.4
6.1±0.3
6.7±0.5
6.1±0.3
6.7±0.5
1.0±0.0
1.2±0.4
1.1±0.3
6.8±0.8
1.1±0.2
1.8±0.4
10
16
16
10
18
15
2.0±0.0
2.0±0.0
2.0±0.0
2.0±0.0
2.0±0.0
2.0±0.0
The spikelets highest along the first panicle were investigated.
into onion bulb epidermal cells by microprojectile
bombardment. Free GFP was used as a negative
control (Figure 6C, D), while the OsMADS3-red
fluorescence protein (RFP) fusion served as a positive control (Sohn et al., 2003). As predicted, the
OsYAB1-GFP fusion protein was restricted to
within the nucleus (Figure 6A). Likewise, coexpression with the OsMADS3-RFP fusion protein (Figure 6B) resulted in co-localization of both
proteins, supporting our hypothesis that OsYAB1
is a nuclear protein.
Figure 6. Subcellular localization by biolistic bombardment of
onion skin cells with GFP as a visible marker. Whereas free
GFP protein was localized in the cytosol (C, D), OsYAB1-GFP
fusion protein was localized in the nucleus (A), as confirmed by
co-bombardment with OsMADS3-RFP fusion protein (B).
Bars¼100 lm.
Discussion
We have reported here our study on the function
of the OsYAB1 gene. Because the primary purpose
of the Arabidopsis YABBY genes is to determine
polarity, we tested whether our OsYAB1 gene had
a similar role in rice. Its ectopic expression in
transgenic rice plants did not alter their polarity.
Instead, the plants bore extra stamens and carpels
in their spikelets. The gene is preferentially expressed in reproductive organs, especially in the
meristems. Because this gene is expressed
throughout all stages of floral organ development,
it may, in fact, be necessary to the maintenance of
this process. Alternatively, the OsYAB1 gene could
be involved in cell division and/or spacing in the
reproductive organs, thereby determine their organ number.
Based on our in-situ data, transcripts were
abundant in the third and fourth whorls of flowers, but without showing polarity. Despite previous genetic and molecular experimental evidence
in Arabidopsis that FIL regulates such floral
homeotic genes as AGAMOUS (AG), APETALA3
(AP3), PISTILLATA (PI), and SUPERMAN
(SUP), together with APETALA 1 (AP1) and/or
LEAFY (LFY), no clear relationship has been
found between polar cell differentiation and
abnormal floral structures, and the effect of ectopic FIL expression has not yet been reported in
Arabidopsis flowers (Chen et al., 1999). Our data
suggest that OsYAB1 plays a role in the development of sexual organs, e.g., stamens and carpels,
rather than in the establishment of cellular polarity. Recently, Yamaguchi et al. (2004) reported
that DL, a rice CRC-like YABBY gene, plays a
key role in carpel specification, floral meristem
determinacy in reproductive organ as well as
midrib formation in leaves, demonstrating that the
expression patterns and functions of the rice
YABBY gene are different from those of Arabidopsis YABBY genes.
Here, we generated transgenic plants that overexpress OsYAB1, one of those genes, to investigate
its function. Unlike the pattern of ectopic expression in Arabidopsis YABBY genes, the development of vegetative organs in transgenic rice plants
did not differ from that of the wild-type plants,
and no abaxialization was present. Instead,
abnormalities, i.e., supernumerary stamens and
carpels, were observed in the floral structures.
141
Similar phenotypic alterations have not been
reported with the Arabidopsis YABBY family
genes. In severe cases, transgenic Arabidopsis
plants that over-express FIL die after forming one
to four rosette leaves; in milder situations, 5 to 10
wrinkled leaves can appear (Sawa et al., 1999b).
Finally, ectopic expression of CRC in that species
does not produce additional carpels despite its
expression pattern being restricted to the carpels
and nectaries of wild-type flowers (Eshed et al.,
1999). Recently, flowers from loss-of-function
mutant alleles of DL showed similar phenotypes
with the OsYAB1 over-expressing flowers. In
addition, ectopic stamens were observed in the
severe dl allele (Yamaguchi et al., 2004). Like
Arabidopsis FIL, the transgenic rice plants
over-expressing DL showed an early death after
producing four to eight leaves. However, leaf
blades of the plants were thickened, and midriblike structure were formed in the lateral regions as
well as the central region by vigorous cell proliferations. The phenotype of OsYAB1 overexpressing flowers may be also explained as the
result of promoted cell proliferation in the inner
whorls of the flowers, resulting in extra stamens
and multiple carpels. When Arabidopsis SUPERMAN (SUP) gene was expressed ectopically in
rice, expansion of the fourth whole was observed
through increased cell proliferation with reduced
stamen numbers. Thus, Nandi et al. (2000) predicted that SUP is a conserved regulator controlling floral cell proliferation.
The difference in expression patterns between
OsYAB1 and Arabidopsis YABBY genes also
suggests that their roles contrast. Our OsYAB1
gene was expressed preferentially in the panicles,
but only weakly in the seedling shoots and flag
leaves. RNA in-situ hybridization results showed
that the OsYAB1 transcripts did not have a polar
expression pattern, as is characteristic of the
Arabidopsis YABBY genes. Different transcript
levels of FIL, YAB2, and YAB3 were detectable in
the cotyledons, leaves, and flowers, but their polar
expression patterns were the same, appearing only
on the abaxial side. CRC expression was also first
detected in abaxial regions of the developing carpels, but was also subsequently found internally, in
cells that would give rise to the placentas. In
addition, CRC transcript was present in cells
adjacent to stamens, which would later form nectaries. In the case of INO, its transcript was
detected only in the abaxial region of the integument (see also Siegfried et al., 1999; Bowman,
2000).
Two cis-acting elements in the 50 regulatory
region of FIL are responsible for its unique
expression pattern (Watanabe and Okada, 2003).
One functions for FIL expression on both the
abaxial and the adaxial sides, the other element
plays a role in repressing expression on the adaxial
side. One possible explanation for the non-polar
pattern seen here with OsYAB1 could be the lack
of the cis-element responsible for repressing
expression in the adaxial region.
Except for the conserved zinc finger and
YABBY domains, OsYAB1 significantly diverges
from the Arabidopsis YABBY proteins. Some
genes in that family, such as CRC and YAB3,
contain many serines and prolines between the zinc
finger motif and the YABBY domain, similar in
composition to the transactivation domains of
several transcription factors (Bowman and Smyth,
1999; Golz and Hudson, 1999). However, the OsYAB1 protein does not possess this property,
again suggesting a different role.
Although Arabidopsis YABBY genes are
strongly implicated in specifying the fate of
abaxial cells in lateral organs, research has shown
that some of these genes also function in floral
organ development. For example, results of genetic epistasis experiments have suggested that
FIL interacts with AP1, LFY, and UNUSUAL
FLORAL ORGANS (UFO) to establish floral
meristem identity (Chen et al., 1999; Sawa et al.,
1999a). This gene is also required for the promotion of flower formation in floral meristems, in
combination with many other genes, including
LFY, UFO, and YAB3. It also appears to influence the correct spacing and number of organs
within a flower (Sawa et al., 1999a). In addition,
FIL regulates floral homeotic genes, such as AG,
AP3, and PI, perhaps through the correct positioning of organs within the expression regions
of those genes (Chen et al., 1999). FIL protein
may also associate with LFY or AP1 to form a
transcriptional complex.
In our study, the overlapping expression pattern
of OsYAB1 with the OsMADS3 and OsMADS15
genes supports the likelihood of a role in rice flower
development. This result may provide a partial
explanation for the phenotypic alterations to
spikelets seen in our transgenic plants. The
142
temporal expression pattern of OsYAB1 during
panicle development, as well as its normal
expression in defective spikelets from FRIZZY
PANICLE, MULTIPLE PISTILS, OsMADS3
knock-out, and OsFOR1 antisense plants, indicates
that OsYAB1 acts from the early stage of spikelet
formation. RNA in-situ hybridization experiments
showed that OsYAB1 transcript was detected
preferentially in the male and female reproductive
organs, a result that coincides with the phenotypic
alterations to the transgenic rice flowers. Taken
together with the overlapping expression patterns
found with floral organ identity genes, it is possible
that OsYAB1 plays a role in C-function, especially
during carpel development.
The CRC gene is expressed preferentially in
carpels and nectaries, and is responsible for carpel
formation. Its ectopic expression induces abaxial
fates in the adaxial regions of leaves, suggesting
that, at least in some contexts, YABBY family
members may substitute for one another (Eshed
et al., 1999). Thus, it will be of interest to investigate the phenotypes of transgenic rice plants that
over-express other rice YABBY genes. Our
OsYAB1 antisense plants showed no phenotypic
abnormalities, perhaps because of the genetic
redundancy that is common with other YABBY
genes. The existence of OsYAB2 supports this
idea. The OsYAB2 transcript is expressed in
developing panicles. Although it remains unclear
which molecular mechanisms are involved in the
phenotypic changes in OsYAB1 over-producers,
the interactions may be complex among OsYAB1
and other floral genes that are responsible for
floral meristem or organ identity. Further research
is needed to reveal the interacting partners with
OsYAB1 and gene targets.
Acknowledgements
We thank Inhwan Hwang for providing the p35SGFP and p35S-RFP vectors, Jong-Jin Han and
Shinyoung Lee for valuable discussion, Sung-Ryul
Kim for sequencing, Jeong-Hee Kim for advice in
constructing phylogenetic tree and Shi-In Kim for
maintaining the transgenic rice plants. We are also
grateful to Priscilla Licht for critical reading of the
manuscript. This work was supported, in part, by
grants from the Crop Functional Genomic Center,
the 21 Century Frontier Program (CG1111 and
CG1114); from the Biogreen 21 Program,
Development Administration; from the
Program, Korea Institute of Science and
nology Evaluation and Planning; from the
Program, Ministry of Education; and
POSCO.
Rural
NRL
TechBK21
from
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