Download Characterization of Two Rice MADS Box Genes That Control

Mol. Cells, Vol. 7, No. 4, pp. 559-566
Characterization of Two Rice MADS Box Genes That
Control Flowering Time
Hong-Gyu Kang, Seonghoe Jang, Jae-Eun Chung, Yong-Gu Chol and Gynbeung An*
Department of Life Science, Pohang University of Science and Technology, Pohang 790-784,
Korea; lGenetics Division, Rural Development Administration, Suwon 441-707, Korea
(Received on May 3, 1997)
Plants contain a variety of the MADS box genes that encode regulatory proteins and play
important roles in both the formation of flower meristem and the determination of floral
organ identity. We have characterized two flower-specific cDNAs from rice, designated
OsMADS7 and OsMADS8. The cDNAs displayed the structure of a typical plant MADS
box gene, which consists of the MADS domain, I region, K domain, and C-terminal region.
These genes were classified as members of the AGL2 gene family based on sequence homology. The OsMADS7 and 8 proteins were most homologous to OM! and FBP2, respectively. The OsMADS7 and 8 transcripts were detectable primarily in carpels and also
weakly in anthers. During flower development, the OsMADS genes started to express at
the young flower stage and the expression continued to the late stage of flower development. The OsMADS7 and 8 genes were mapped on the long arms of the chromosome
8 and 9, respectively. To study the functions of the genes, the eDNA clones were expressed
ectopically using the CaMV 35S promoter in a heterologous tobacco plant system.
Transgenic plants expressing the OsMADS genes exhibited the phenotype of early flowering and dwarfism. The strength of the phenotypes was proportional to the levels of
transgene expression and the phenotypes were co-inherited with the kanamycin resistant
gene to the next generation. These results indicate that OsMADS7 and 8 are structurally
related to the AGL2 family and are involved in controlling flowering time.
Floral initiation is controlled by several factors including photoperiod, cold treatment, hormones, and
nutrients (Coen, 1991; Gasser, 1991). Physiological
studies have demonstrated that vegetative tissues are
the site for the signal perception and for generation
of chemicals that cause the transition from vegetative
growth to flowering (Lang, 1965; Zeevaart, 1984).
Genetic analysis revealed that there are several types
of mutants that alter flowering time. In Arabidopsis,
there are at least two groups of mutants based on
their response to photoperiod and vernalization (Martinez-Zapater et al., 1994). These phenotypes suggest
that there are multiple pathways that lead to flowering.
Studies on mutants that interfere with normal flower development have provided some information on
the mechanisms of the development. This led to the
information that there are at least two genes needed
for induction of flower development: LEAFY (LFY)
and APETALAI (API) genes in Arabidopsis (Weigel,
1995), and FLORICAULA (FLO) and SQUAMOSA
(SQUA) genes in Antirhinnus (Bradley et at., 1993).
Cloning and analysis of these genes revealed that the
LFY and FLO genes are homologs and encode pro-
* To whom correspondence should be addressed.
teins with a proline-rich region at its N-terminus and
a highly acidic central region which are features of
certain types of transcription factors (Coen et aI.,
1990; Weigel et al., 1992). The API and SQUA gene
products are transcription factors that contain a conserved MADS box sequence (Huijser et al., 1992;
Mandel et at., 1992). MADS box containing genes
were isolated from several plant species and are
known to play important roles in plant development,
especially flower development. Arabidopsis homeotic
genes, AGAMOUS (AG), PISTlLATA (PI), and APETALA3 (AP3) are members of the MADS box gene
family (Goto and Meyerowitz, 1994; Jack et al. , 1992;
Yanofsky et al., 1990). Similar homeotic genes from
A. majus, PLENA (PLE), GLOBOSA (GLO) , and DEFICIENS A (DEFA) , are also members of the MADS
box genes (Bradley et aI. , 1993; Sommer et al., 1990;
Trobner et al. , 1992). Characterization of these gene
products showed that the conserved MADS box
domain is for sequence-specific DNA binding, dimerization, and attraction of secondary factors (Pellegrini et aI., 1995). The DNA sequences with which
the MADS box domains interact are the consensus
Accession numbers: OsMADS7, U78891; OsMADS8, U
78892.
©
1997 The Korean Society for Molecular Biology
560
Rice MADS Genes Controlling Flowering Time
binding sites, CCA!T6GG (Huang et aI. , 1993; 1995;
Pollock and Treisman, 1991). In addition to the
MADS box domain, the plant MADS box proteins include the K domain, a second conserved region carrying 65-70 amino acid residues. The K domain was
named due to the structural resemblance to the coiled
coil domain of keratin (Ma et ai., 1991), and has
been suggested to be related to protein-protein interactions (Pnueli et aI., 1991). Similar MADS box
genes have also been studied in other plants including tomato, Brassica napus, tobacco, petunia, maize,
and rice (Thei~en and Saedler,1995). Within the past
few years, a considerable number of plant MADS
box genes that deviate from the functions of the typical meristem identity and organ identity genes have
been identified. These genes are involved in the control of ovule development (Angenent et al., 1995),
vegetative growth (Mandel et al., 1994), root development (Rounseley et al. , 1995), embryogenesis
(Heck et al. , 1995) or symbiotic induction (Heard
and Dunn, 1995).
There are a large number of MADS box genes in
each plant species. In maize, at least 50 different
MADS box genes consist of a multigene family and
these genes are dispersed in the plant genome (Fischer et al., 1995; Mena et al., 1995). The MADS box
multigene family can be divided into several subfamilies according to their primary sequences, expression patterns and functions (Thei~en and Saedler,
1995). We have previously reported the isolation of
five MADS box genes of rice: OsMADS1, OsMADS2,
OsMADS3, OsMADS4, and OsMADS5. Sequence similarity to other MADS box genes indicated that
OsMADSl and OsMADS5 belong to the AGL2 family
(Chung et al., 1994; Kang and An, 1997), OsMADS2
and OsMADS4 to the GLOBOSA family (Chung et al. ,
1995), and OsMADS3 to the AG family (Kang et al.,
1995). Functional analysis by ectopic expression in a
heterologous tobacco system indicated that OsMADSl
and OsMADS5 are involved in controlling flowering
time and OsMADS3 is important for anther development. In this paper, we report the isolation and
characterization of two additional MADS box genes
of rice that belong to the AGL2 family and induce an
early flowering in tobacco plants.
Materials and Methods
Bacterial strains, plant materials, and plant
transformation
Escherichia coli JM 83 was used as the recipient
for routine cloning experiments. Agrobacterium tumefaciens LBA4404 (Hoekema et al., 1983) containing
the Ach5 chromosomal background and a disarmed
helper-Ti plasmid pAL4404 was used for transformation of tobacco plants (N. tabacum L. cv. Xanthi)
by the cocultivation method (An et al. , 1988). Transgenic plants were maintained under greenhouse conditions. Rice (Oryza sativa L. cv. M201) plants were
Mol. Cells
grown in a growth chamber at 29°C with a 10.5 h
day cycle.
Library screening and sequence analysis
cDNA libraries were constructed from mRNAs prepared from rice flowers at floral primordia and young
flowers (length of the panicle was below 1 cm). Hybridization was performed with 105 plaques using a
32P-labeled probe of the OsMADSl coding region.
The cDNA insert was rescued in vivo using an f1
helper phage, R408 (Stratagene). Both strands of the
cDNA were sequenced by the dideoxynucleotide
chain termination method using a double-strand DNA
as a template (Sanger et al., 1977). Protein sequence
similarity was analyzed by the IG Suite software
package (Intelligenetics Co., Mountain View, CA)
and the NCBI non-redundant protein database on the
international network.
DNA and RNA blot analyses
Total genomic DNA was isolated by the CTAB
(cetyltrimethylammonium bromide) method from twoweek-old rice seedlings grown hydroponically (Rogers and Bendich, 1988). Eight Ilg of total DNA
were digested with the appropriate restriction enzymes, separated on a 0.7% agarose gel, blotted onto
a 'nylon membrane, and hybridized with a 32P-labeled
probe for 16 h at 65 °C in a solution containing 6 x
SSC and 0.2% BLOTTO (Sambrook et al., 1989).
Mter hybridization, the blot was washed with a solution containing 2 x SSC and 0.5% SDS for 20 min
at 65°C, followed by a wash with a solution of 0.1 x
SSC and 0.1 % SDS for 15 min at the same temperature. Total RNA was isolated by the guanidium
thiocyanate method (Sambrook et al. , 1989). Leaf
and root samples were harvested from the two-weekold seedlings. Floral organ samples were obtained by
dissecting late vacuolated stage flowers under a dissecting microscope. Twenty-five mg of total RNA
was fractionated on a 1.3% agarose gel as described
previously (Sambrook et al., 1989). After RNA transfer onto a nylon membrane, the blot was hybridized
in a solution containing 0.5 M NaP0 4 (PH 7.2), 1
mM EDTA, 1% BSA, and 7% SDS for 20 h at 60°C
(Church and Gilbert, 1984). After hybridization, the
blot was washed twice with a solution containing
0.1 x SSPE and 0.1 % SDS for 5 min at room temperature followed by two washes of the same solution at 60°C for 15 min.
Mapping procedures
An F11 recombinant inbred population conslstmg
of 164 lines derived from a cross between Milyang 23
and Gihobyeo was used to construct a molecular map.
Three-week old leaf tissue was harvested from over
one hundred seedlings of each F11 line and bulked
for DNA extraction as described (Cho et aI., 1997).
DNA was digested with 8 restriction enzymes
(BamHI, DraI, EcoRI, HindIII, EcoRV, Scal, Xbal,
KpnI) and 8
~g
per lane was used to make mapping
filters. DNA blotting and hybridization were performed as previously described (Cho et aI. , 1994).
Linkage analysis was performed using Mapmaker
Version 3.0 (Lander et aI. , 1987) on a Macintosh
Power PC 8100/80A V. Map units (cM) were derived
using the Kosambi function (Kosambi, 1944).
Results
Isolation of rice cDNA clones encoding MADS box
proteins
We have isolated two cDNA clones by screening a
A Zap II cDNA library prepared from rice floral primordia using OsMADSl cDNA as a probe (Chung et
al. , 1994). The clones were designated OsMADS7
and OsMADS8. DNA sequence analysis showed that
th e OsMADS7 and 8 clones are 1060 and 1259 nucleotides long and encode putative proteins of 249
and 248 amino acid residues, respectively (Figs. 1
and 2). The 5' UTR of OsMADS8 cDNA contains 8
repeats of the GGA sequence and that of OsMADS7
cDNA contains 6 repeats of the GGT sequence, so
called a rnicrosatellite (Browne and Litt, 1991; Stallings, 1992). Such repeat sequences were previously
observed fro m the rice MADS-box gene family
(Chung et al. , 1994; Kang and An, 1997).
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Figure 2. Nucleotide and deduced amino acid sequences
of the OsMADS8 eDNA. The MADS box and K box regions are underlined. The positions of nucleotides and amino acids are shown on the left and right, respectively. The
double underl ine is the NheI site, which was used to generate the gene-specific probe of the 230 bp fragment located at the 3' region of the eDNA.
28
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Hong-Gyu Kang et al.
Vol. 7 (1997)
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12 8
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Figure 1. Nucleotide and deduced amino acid sequences
of the OsMADS7 eDNA. The MADS box and K box regions are underlined. The positions of nucleotides and amino acids are shown on the left and right, respectively. The
double underline is the Pstl site, which was used to generate the gene-specific probe of the 280 bp fragment located at the 3' region of the eDNA.
The MADS box domain of the cDNA clone is lond
th
cated between the 2 and 57 amino acids of each
protein (Figs. 1 and 2). This region is the most conserved region as observed from other MADS box
genes. The second conserved domain, the K box, is
located between the residues 95 and 160 in both
OsMADS7 and OsMADS8 (Figs. 1 and 2) . The
genes contain two variable regions, the I region
between the MADS and K boxes, and the C region
downstream from the K box (Purugganan et aI. ,
1995). These observations suggest that OsMADS7
and 8 encode proteins showing the typical structure
of the plant MADS box gene family. Based on amino
acid sequence similarity of the entire coding region,
the OsMADS7 and 8 proteins can be grouped into
the AGL2 family which includes AGL2, AGL4 and
AGL6 of Arabidopsis (Ma et aI. , 1991), FBP2 of
petunia (Angenent et al. , 1994), ZAG3 and ZAG5 of
maize (Men a et aI., 1995), TM5 of tomato (Pnueli et
aI. , 1994b), OMI of orchid (Lu et aI. , 1993), and
OsMADSl and 5 of rice (Chung et aI. , 1994; Kang
and An, 1997). Among these genes, the OsMADS7
and 8 proteins were most homologous to OMI (61 %
and 65%, respectively) and FBP2 (60% and 64%,
respectively). Both of the OsMADS proteins were
50% identical to OsMADSl.
Rice MADS Genes Controlling Flowering Time
562
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Figure 3. Alignments of OsMADS7 and OsMSDS8 proteins with other MADS box proteins. The MADS box (A)
K box (B) and C-termiral end (C) regions were aligned by
introducing gaps (points) for the maximum sequence
homology. Highlights indicate conservative sequences. (D)
shows the structure of MADS box proteins. M, MADS
box region; I, I region; K, K box region; C, C-terminal
region; CE, C terminal end region.
Alignment of the OsMADS7 and 8 proteins with
other members of the AGL2 family showed that the
MADS box, K box and C terminal end regions share
significant sequence homologies (Fig. 3). The MADS
box region of both of the proteins is 100% identical
to that of AGL2 and FBP2. The MADS box sequences of OsMADS7 and 8 share at least 89% identity to that of other AGL2 proteins. The sequence
homology in the K box region is lower compared to
the MADS box region, but still significant. The regions of OsMADS7 and 8 are at least 43% identical
to other members in the family, whereas the homology is much lower with the distantly related MADS
box proteins, such as AG, AP3 , and PI. The sequence homology at the C terminal end is much lower. However, there are two blocks of highly conserved regions at the end of proteins and these AGL2
specific sequences are not found in other MADS box
proteins.
Figure 4. DNA blot analyses of rice genomic DNA with
OsMADS7 or OsMADS8. Rice genomic DNA was digested
with EcoRI (E), HindIII (H) or Pst! (P), fractionated on a
0.8% agarose gel, and hybridized with the 280 bp EcoRIPst! fragment of OsMADS7 (A) and the 230 bp EcoRINheI fragment of OsMADS8 (B), respectively. The positions of HindlII digested lambda DNA size markers are
indicated on the right side of the figure .
DNA and RNA blot analyses
It is well established that there are a large number
of MADS box genes in the rice genome (Chung et
ai. , 1994). Genomic DNA blot analyses were conducted to identify the region that does not cross hybridize with other MADS box genes (Fig. 4). It was
observed that the 280 bp PstI-EcoRI fragment which
is located at the C-terminal region of OsMADS7 hybridized to a single DNA fragment. Likewise, the 220
bp NheI-EcoRI fragment of OsMADS8 is shown to
be the gene specific region.
RNA blot analysis was conducted using the gene
specific probes. The results show that both of the
OsMADS7 and 8 transcripts were detectable primarily
in carpels and also weakly in anthers. However, the
transcripts were not detectable in the palea!lemma or
vegetative organs (Fig. SA). This expression pattern
was similar to that of OsMADS3 and OsMADS4
(Chung et ai. , 1995; Kang et ai., 1995). During flower development, the OsMADS7 gene was strongly expressed at the young flower stage and the expression
was gradually decreased as the flowers further developed to the mature flower stage, whereas the
OsMADS8 expression was weak at the young flower
stage and the expression was gradually increased as
the flowers developed (Fig. 5B).
Chromosomal mapping of the OsMADS genes
An Fll recombinant inbred population of rice was
Vol. 7 (1997)
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MADS8
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Figure 5. RNA blot analyses of OsMADS7 or OsMADS8
transcript in rice. A) Expression patterns of OsMADS7 or
OsMADS8 in rice organs. Ten Ilg of total RNAs isolated
from leaves (L) and roots (R) of two-week-old seedlings,
and paleas/lemmas (P), anthers (A), and carpels (C) of late
vacuolated-stage flowers were hybridized with the genespecific probes. B) Temporal expression pattern of
OsMADS7 or OsMADS8 during flower development. Twenty-five Ilg of total RNAs isolated from rice flowers at different developmental stages were used for the detection of
the OsMADS gene expression. Samples 1, young flowers at
the panicle size 1 to 5 cm; 2, flowers at the early vacuolated
pollen stage; 3, flowers at the late vacuolated pollen stage.
Ethidium bromide staining of 25S and 18S rRNAs were
shown to demonstrate equal amounts of RNA loading.
used to locate the OsMADS genes on a genetic map.
The 3'-end of the DNA fragments which were shown
to be unique to each OsMADS gene were used. These
experiments revealed that OsMADS7 is located on the
long arm of chromosome 8 and OsMADS8 is on the
long arm of chromosome 9 (Fig 7). We also mapped
three additional rice MADS box genes, OsMADS2,
OsMADS3, and OsMADS5 that were previously
characterized (Chung et aI., 1995; Kang et at. , 1995;
Kang and An, 1997). It was shown that OsMADS2 is
a member of the GLOBOSA family . This gene is located on the long arm of the chromosome 1 (Fig. 6).
OsMADS3 is a rice homolog of Arabidopsis
AGAMOUS and is located on the short arm of chromosome 1. OsMADS5 is located on the long arm of
chromosome 6.
$.9 -
'-7
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RCt»
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MADS7
RZ70A
11.7 -
20.9 RO)11
).9-
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~
11.0 109
....,
MADS2
Figure 6. Genetic maps of the OsMADS genes. The locations of OsMADS genes along with RFLP markers (RG
and G), cDNA markers (RZ and C), and microsatellite
markers (RM) are indicated. Map distance is given in cM
on the left of each chromosome. Dark bars represt<nt the
centromere regions.
shorter and bloomed earlier than the control plants
which were transformed with the Ti plasmid vector
alone while others showed a normal growth . The
RNA blot analyses were conducted to investigate the
expression levels of the transgenes (Fig. 7). In order
to minimize variations due to the development stage,
young leaves at the anthesis of the first flower were
used for the isolation of RNA. These analyses revealed that plants showing the early flowering phenotype expressed higher levels of the transgene compared with transgenic plants exhibiting weak or no
phenotype. Transgenic lines, OsMADS7-5, - 9, and
- 10, and transgenic lines, OsMADS8-2 and - 6, ac1
2
3
4
5
6
7
8
9
10
1
2
3
4
5
6
7
8
9
10
A
Ectopic expression
The functional roles of the two rice MADS box
genes were studied using tobacco plants as a heterologous expression system. The eDNA clones were
placed under the control of CaMV 35S promoter and
transcript 7 terminator using binary vector pGA7 48
(An et ai., 1988). The chimeric molecules were transferred into tobacco plants using the kanamycin resistant marker and Agrobacterium-mediated Ti plasmid
vector system. Ten independent TI transgenic plants
were regenerated to avoid any artifact. Results showed that some of the primary transgenic plants were
B
Figure 7. RNA blot analyses of transgenic tobacco plants
expressing the OsMADS7 (A) or OsMADS8 (B) transcript.
Ten Ilg of total RNAs isolated from young leaves of the
transgenic plants were hybridized with the gene-specific
probe. The numbers indicate independently transformed
plants.
564
Rice MADS Genes Controlling Flowering Time
Figure 8. Phenotypes of transgenic plants expressing
OsMADS7 (A) or OsMADS8 (B). The two left plants are
transgenic lines at the T2 generation and the two right
plants are controls, the wild-type Xanthi tobacco.
cumulated higher levels of the transgene transcript and
flowered earlier. Transgenic lines which displayed the
most severe phenotype were selected to examine the inheritance of the characteristics. The result showed that
the phenotypes were co-inherited with the kanamycin
resistant gene to the next generation (Fig. 8). Both of
the transgenic plant lines, OsMADS7-1O and OsMADS
8-6, flowered 9 days earlier and were significantly shorter than the controls.
Discussion
We isolated two additional rice MADS box genes
that are probably involved in controlling flowering
time. The deduced amino acid sequences of the gene
products showed a high homology to the AGL2 family proteins. The homology was extensive covering
all the proteins. It was observed that the AGL2 family proteins could be further divided into several subgroups depending on the protein sequence similarity
in the K box and the two variable regions (Thei~en
and Saedler, 1995). Our results in Figure 3 show that
OsMADS7 and OsMADS8 belong to the FBP2 sub-
Mol. Cells
family. The sequence identity suggests that they share
a similar biological function . The roles of FBP2 were
investigated by the co-suppression approach (Angenent et ai., 1994). When the FBP2 expression was
suppressed in petunia flowers, it resulted in aberrant
flowers with modified whorl two, three, and four
organs. The flower possessed a green corolla,
petaloid stamens, and dramatically altered carpel structure. Therefore, it seems that FBP2 is involved in the
determination of the central parts of the generative
meristem. Similar effects on development of the inner
three whorls have also been observed by antisense
RNA approach of the tomato MADS box gene, TM5
(Plunei et al., 1994b). The role of OsMADS1 and 5
was studied by ectopically expressing the cDNA
clones in heterologous tobacco plants (Chung et ai.,
1994; Kang and An, 1997). Transgenic plants overexpressing OsMADS1 or 5 exhibited an early flowering and dwarf phenotypes, suggesting that the
genes are involved in controlling the flowering time.
No morphological alterations of the floral organs
were observed. These observations suggest that the
FBP2 and TM5 genes function differently from the
OsMADS1 gene. It is interesting to observe that the
MADS box proteins of OsMADSl, 5, 7, and 8, and
API that caused an early flowering consist of 248-257
amino acids whereas FBP2 and TM5 proteins consist
of 224 amino acids. It is possible that the additional
amino acids present in the OsMADS genes are responsible for controlling the flowering time. Further studies are needed to understand the role of the AGL2
family .
RNA blot analyses showed that the OsMADS7 and
8 genes were expressed specifically in flowers and
such a specificity is coincided with that of the AGL2
family genes. This indicates that the genes of the
AGL2 family function primarily during flower development. The expression of the OsMADS genes all
started at the early stage of flower development and
extended into later stages of flower development, indicating that the OsMADS genes play critical roles
during the early stage and continue to function as the
flower further develops. Such expression patterns
were also observed from other AGL2 members, including AGL2 and 4, FBP2, TM5, and OsMADS1
and 5 (Angenent et al., 1992; Chung et al., 1994;
Kang and An, 1997; Ma et ai., 1991; Prueli et al.,
1991). However, not all the AGL2 family members
are early genes. The OM1 transcript was detectable
only after flower organs fully developed (Lu et aI.,
1993). The FBP2 and TM5 genes were expressed in
the whorls 2, 3, and 4 (Angenent et aI. , 1992; Prueli
et aI. , 1991). Unlike most dicots, rice flowers contain
a single perianth, pale/lemma, which is closer in
resemblance to whorl sepal than petal. The palea/lemrna contains chlorophyll and remains attached to the
mature seeds. Therefore, it is expected that expression of FBP2 homo logs in dicots should be restricted in sepals and petals. The OsMADS7 and 8
Vol. 7 (1997)
Hong-Gyu Kang et al.
genes were expressed in the two inner whorls, coinciding with the expected expression pattern. This indicates that a high amino acid homology between
MADS box genes does not necessarily coincide in
their expression patterns.
The OsMADS genes were mapped on rice chromosomes. The results showed OsMADS7 and 8 are located on chromosomes 8 and 9, respectively. It was
previously reported that the EF-1 gene for duration to
flowering time in rice is located on chromosome 10,
and the Se genes for photoperiod sensitivity are positioned on chromosome 6 or 7 (Khush and Kinoshita,
1991). Therefore, it is evident that OsMADS5 , 7, and
8 genes are not linked to the previously mapped
markers that are involved in controlling the timing of
flowering . The relationship to other heading time
genes, such as E-1, E-2, E-3, 1[-1 , and 1[-2, can be
resolved when these genes are mapped. We have included map information of the previously studied
OsMADS genes. The OsMADS2 gene that is a member of the GLOBOSA family is located near the RG
109 and the EstI-2 on chromosome 1. It was previously reported that the RG109 and the Estl-2 markers are tightly linked to the semidwarf gene, sd-1 ,
which is important for controlling culm length and
flowering time (Causse et aI. , 1994; Cho et aI. , 1994).
The OsMADS5 gene is mapped on chromosome 6, about 20-30 cM away from the photoperiod sensitivity
genes, Se-1 and Se-3 (Causse et aI. , 1994).
To elucidate the functions of the rice MADS box
genes, we have generated transgenic tobacco plants expressing a chimeric fusion between the CaMV 35S promoter and the OsMADS cDNA. Both OsMADS genes
caused the phenotype of early flowering and dwarfism
when the genes were strongly expressed in transgenic
plants. We have previously identified two early flowering genes, OsMADS1 and 5, from rice. Therefore, it
appears that there are at least four genes in rice which
are involved in controlling flowering time. It will be interesting to study whether expression of the OsMADS
genes are differently controlled by different environmental or developmental factors.
Acknowledgments
This study was supported by grants from the Korean
Science and Engineering Foundation (96-0401-06-01-3)
and from the Basic Science Research Institute, Pohang
University of Science and Technology (96F120).
We thank Kyungsook An and Miyoung Lee for
maintaining transgenic tobacco, Chahm An for critical reading of the manuscript and Drs Suns an R.
M~Couch and Takuji S ~saki for providing rice molecular markers.
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