DEVELOPMENTAL GENETICS 25:237–244 (1999)
OsMADS13, A Novel Rice MADS-Box Gene Expressed
During Ovule Development
ZENAIDA P. LOPEZ-DEE,1 PETER WITTICH,2 M. ENRICO PÈ,1 DIANA RIGOLA,1 ILARIA DEL BUONO,1
MIRELLA SARI GORLA,1 MARTIN M. KATER,1 AND LUCIA COLOMBO1*
1Department of Genetics and Microbiology, University of Milan, Milan, Italy
2Department of Plant Cytology and Morphology, Wageningen Agricultural University, Wageningen, The Netherlands
ABSTRACT MADS-box genes have been shown
to play a major role in defining plant architecture.
Recently, several MADS-box genes have been reported
that are highly expressed in the ovule. However, only for
the Petunia genes FBP7 and FBP11 has a function in
defining ovule identity been shown. We have isolated a
rice MADS-box gene named OsMADS13. Expression
analysis has shown that this gene is highly expressed in
developing ovules. In order to facilitate a detailed
characterization of rice ovule-expressed genes, a compre-
hensive morphological description of ovule development
in rice has been performed. The predicted amino acid
sequence of OsMADS13 shows significant homology
with ZAG2, a maize MADS-box gene, which is also
expressed mainly in the ovule. Mapping of the gene in
the rice genome showed that it is located on chromo-
some 12, which is syntenic to two maize regions where
ZAG2 and its paralogous gene ZMM1 have been
mapped. Our results suggest that OsMADS13 is the
ortholog of ZAG2 and ZMM1 and might play a role in
rice ovule and seed development. Dev. Genet. 25:237–
244, 1999. r 1999 Wiley-Liss, Inc.
Key words: MADS-box genes; rice; ovule develop-
ment; synteny; OsMADS13; ZAG2; FBP11
INTRODUCTION
The ovule is the most important part of the female
reproductive organ. It is the site of megasporogenesis,
megagametogenesis, and fertilization and, as the precur-
sor of the seed, it plays a major role in the reproductive
cycle of the plant. In angiosperms, ovules are part of the
gynoecium, which consists of one or more carpels. Ovule
development has been studied in a wide variety of
flowering plants [Bouman, 1984; Reiser and Fischer,
1993; van Tieghem, 1898; Warming, 1878; Willemse
and van Went, 1984]. However, because Arabidopsis is
considered to be the model species for studying develop-
mental processes in dicots, most such research is cur-
rently done on this species. A detailed morphological
study of Arabidopsis ovule development was been done
[Schneitz et al., 1995] in order to gain a better under-
r 1999 WILEY-LISS, INC.
standing of ovule development and to serve as a guide
for further genetic and molecular analysis.
Several Arabidopsis mutants have been identified
that affect ovule development [for a review, see Schneitz
et al., 1998a]. Almost all of them show defects in
integument development, i.e., mutations in the
AINTEGUMENTA (ANT ) [Elliott et al., 1996; Klucher
et al., 1996] and HUELLENLOS (HLL) [Schneitz et al.,
1998b] loci, which lead to almost complete absence of
integuments. Mutations in BELL1 (BEL1) [Modrusan
et al., 1994; Robinson-Beers et al., 1992; Schneitz et al.,
1997] result in the arrest of inner integument develop-
ment, whereas in INNER NO OUTER (INO) mutant,
the development of the outer integument is blocked.
These studies have led to the postulation of models for
the genetic control of ovule development [Angenent and
Colombo, 1996; Baker et al., 1997; Schneitz et al., 1998a].
In Arabidopsis, genes controlling ovule identity have
not yet been characterized. However, in Petunia, two
genes, FLORAL BINDING PROTEIN7 (FBP7) and
FBP11, have been identified that control ovule identity
[Angenent et al., 1995; Colombo et al., 1995]. Both
genes, which share 90% identity and are probably
redundant, encode transcription factors of the MADS-
box family. FBP7 and FBP11 are expressed in the
center of the gynoecium before ovule primordia are
visible; at later stages of development, their expression
can only be detected in the ovules. Simultaneous inhibi-
tion of these genes by cosuppression resulted in trans-
formation of ovules into carpelloid structures [Ange-
nent et al., 1995], showing that these two genes are
necessary to determine ovule identity. Furthermore,
ectopic expression of FBP7 or FBP11 in Petunia in-
Contract grant sponsor: EU-BIOTECH; Contract grant number: BIO4-
CT97-2217; Contract grant sponsor: NATO; Contract grant number:
CRG.CRG 973222. This project was supported by the EU-BIOTECH
project number BIO4-CT97-2217.
*Correspondence to: Lucia Colombo, Department of Genetics and
Microbiology, University of Milan, via Celoria 26, 20133 Milan, Italy.
E-mail: lucia.colombo@unimi.it
Received 10 March 1999; Accepted 1 June 1999
238 LOPEZ-DEE ET AL.
duced the formation of ovules on sepals and petals
suggesting that FBP7 and FBP11 are sufficient to
induce ovule development [Colombo et al., 1995]. In
Arabidopsis, a sequence homologous to FBP11 has been
isolated, namely AGL11 [Rounsley et al., 1995]. How-
ever, the involvement of AGL11 in determining ovule
identity has not been shown so far.
In monocots, two of the few cloned MADS-box genes
expressed during ovule development are the ZAG2 and
ZMM1 genes of maize [Schmidt, 1993; Theissen et al.,
1995]. ZAG2 expression was first detected in carpel
primordia and its expression has been found to persist
until maturation of the ovules. Transcripts of ZAG2
were also detected in cells lining the inner face of the
carpels and in the silks [Schmidt et al., 1993]. ZMM1 is
very similar to ZAG2, and it is likely that the two genes
have been originated by allotetraploidization [Theissen
et al., 1995].
This article describes the isolation and characteriza-
tion of a novel MADS-box gene of rice, namely Os-
MADS13, which is specifically expressed during ovule
development. Genetic mapping data show that the
synteny between the rice and maize genomes makes it
possible to identify the corresponding genes in these
two monocot species. In order to produce a developmen-
tal calendar that will be useful as a reference for ovule
development studies in rice, we present a detailed
description of the developmental events during rice
embryo sac development.
MATERIALS AND METHODS
Plant Material
Rice (Oryza sativa L., var. Taipei 309) inflorescence,
leaves, roots, and kernels at different stages of develop-
ment were collected from the field at the Ente Nazion-
ale Riso, Castel d’Agogna (PV), and from the botanical
garden of the University of Milan.
Isolation of OsMADS13
The rice ovule and young kernel cDNA libraries were
constructed using the HybriZap cDNA Gigapack Clon-
ing Kit (Stratagene), according to the manufacturer’s
protocol. Approximately 30,000 plaques of each library
were screened with MADS-box regions of the rice gene
OsMADS45, the maize gene ZAG2, and the petunia
gene FBP11. Probes were labeled using the random
priming DNA labeling kit (Boehringer Mannheim).
Pre-hybridization and hybridization were performed as
described [Kater et al., 1998].
RNA Blot Analysis
Total RNA was isolated from leaves, roots, ovules,
developing inflorescence, and kernels. RNA extraction
was performed following the protocol proposed by Kater
et al. [1998]. A total of 10 µg of total RNA was loaded on
a 2% agarose gel as already described by Kater et al.,
1998].
In Situ Hybridization
Developing inflorescences of wild-type rice plants
were fixed and embedded in paraffin, according to the
protocol of Cañas et al. [1993]. Digoxygenin-labeled
antisense RNA probes were generated by in vitro
transcription according to the instructions provided
with the In Vitro Transcription Kit (Boehringer Mann-
heim). Hybridization and immunological detection were
performed as described by Cañas et al. [1993]. Sections
were photographed with a Zeiss Axiophot microscope.
Genomic Mapping
Gene specific probes lacking the conserved MADS-
box coding region were produced for OsMADS13 and for
the maize gene ZAG2 [Schmidt et al., 1993] from cDNA
clones. The two genes were mapped as restriction
fragment length polymorphisms (RFLPs) onto the high-
density rice genetic linkage map of Harushima et al.
[1998], following a previously described protocol [Kurata
et al., 1994].
Light Microscopy
For Technovit embedding, materials were fixed for
2 h in 5% glutaraldehyde and 0.1 M phosphate buffer, at
pH 7.2. This was followed by washing four times in
buffer, and then twice in water for 15 min each wash.
Materials were then dehydrated in an ethanol series
(10%, 30%, 50%, 70%, and 90%) for 1 h each. Final
dehydration in 100% ethanol was done twice for 15 min
each. Infiltration and polymerization in Technovit (Kul-
zer) was done according to the manufacturer’s instruc-
tions; 3- to 5-µm-thick sections were stained in 1%
toluidine blue and mounted in distilled water. Selected
sections were photographed with a Panasonic digital
camera and processed using the software AcQuis (Syn-
optics) and Adobe Photoshop 5.0.
RESULTS
Ovule Development in Wild-Type Rice Flowers
The rice ovule is bitegmic, exhibiting monosporic
megasporogenesis and Polygonum-type embryo sac de-
velopment. From an original position of about 70° from
the vertical axis, the developing ovule curves down-
ward through approximately 90°. The sequence of
developmental events that occur in the rice ovule can be
divided into nine distinct stages (Table 1). A range of
inflorescence lengths is given as a reference since
inflorescence length varies between individual plants.
The ovule primordium develops from the inner layer of
the floral apex. It is initiated early in the development
of the spikelet, before the inflorescence reaches a length
of 1 cm. We assign this developmental event as ovule
stage 1 (Ov1). Consisting of a round mass of actively
dividing cells, the main bulk of the primordium be-
comes the nucellus. Near its base and on opposite sides,
the integument primordia form (Fig. 1B). Outer and
inner integuments differentiate from these primordia,
 MADS-BOX GENES AND RICE DEVELOPMENT
TABLE 1. Ovule Development in Rice, var. Taipei 309
Range of
Inflorescence-
Morphology & Stage Length
Developmental Events (cm)
Ov1 Ovule primordium develops as a round ⬍1.0
mass of actively dividing cells.
Ov2 Megasporogenesis starts with the for- 1–3
mation of the archespore from one of
the subepidermal cells in the
nucellus. The archespore is distin-
guished by its larger size. Integument
primordia are initiated on both sides
of the ovule primordium, followed by
differentiation firstly into the outer
integument and later into the inner
integument. Funiculus develops on
the adaxial side of the ovary.
Ov3 The archespore expands and differenti- 3–8
ates into the megaspore mother cell.
The megaspore mother cell is polar-
ized with its nucleus at the micro-
pylar pole.
Ov4 Meiotic division of the megaspore 6–10
mother cell results in a linear tetrad
of megaspores. The inner integument
fully encloses the nucellus, except for
the area where the micropyle forms.
Ov5 Micropylar megaspores degenerate and 10–11
the functional megaspore enlarges.
The ovule has rotated approximately
60° from its original position.
Ov6 The developing embryo sac is at the 12–14
two-nucleate megagametophyte
stage. Degenerated megaspores are
still visible.
Ov7 A large central vacuole develops 14–15
between the two nuclei of the mega-
gametophyte, followed by mitosis of
both nuclei, resulting in the four-
nucleate stage.
Ov8 Formation of the eight-nucleate mega- 16–18
gametophyte, followed by migration
of the nuclei and cellularization. The
ovule has rotated approximately 90°
from its original position.
Ov9 Embryo sac maturation: polarization of 18–24
the egg cell and synergids, fusion of
the polar nuclei, and mitotic division
of the antipodals to yield as many as
eight cells. A single layer of four to
five large nucellus cells borders the
micropyle.
with the outer integument differentiating earlier than
the inner integument. Whereas the outer integument
develops from both epidermal and subepidermal cells,
the inner integument originates from the L1 layer of
the primordium. Development of the integuments on
opposite sides is not synchronous. Two cell layers form
the inner integument, while up to three layers comprise
the outer integument. The inner integument com-
pletely envelops the nucellus as megasporogenesis pro-
ceeds. Growth of the outer integument, however, does
not keep up with the downward curvature of the ovule.
239
Hence, the basal portion of the mature ovule is covered
by both integuments, but the abaxial side of the ovule is
enclosed only by the inner integument (Fig. 1I). The
funiculus develops on the adaxial side of the ovule. A
single vascular strand runs along the funiculus.
Megasporogenesis and Megagametogenesis
Below the nucellus epidermis, a large cell develops
into the archespore (Fig. 1A and B) stage Ov2. Subse-
quently, it enlarges, becomes polarized, and differenti-
ates into the megaspore mother cell (Ov3). As the
megaspore mother cell expands, its cytoplasm becomes
less dense than those of the surrounding nucellus cells.
The megaspore mother cell is characterized by a promi-
nent nucleus that occupies the micropylar half of the
cell (Fig. 1C).
Megasporogenesis soon follows with the meiotic divi-
sion of the megaspore mother cell (Ov4) and a linear
tetrad of megaspores (Fig. 1D) is produced. The three
megaspores at the micropylar pole soon degenerate,
leaving the chalazal megaspore (panel 4, Fig. 1D) as the
functional megaspore. The functional megaspore en-
larges (Ov5) and develops a very large nucleolus (Fig.
1E,F). It undergoes the first mitotic division resulting
in the two-nucleate stage (Ov6) of female gametophyte
development (Fig. 1G). As the female gametophyte
develops, a large vacuole forms at the chalazal pole of
the megagametophyte. Remnants of the degenerated
megaspores are still present until the early four-
nucleate stage. The female gametophyte enters the
four-nucleate stage (Ov7), with a large central vacuole
forming between the two nuclei (data not shown).
Subsequently, the nuclei divide a second time. The
resulting daughter nuclei at the micropylar end are
initially horizontally aligned but are soon realigned
along the micropylar-chalazal axis. The third mitotic
division occurs rapidly, resulting in an eight-nucleate
megagametophyte. Nuclear migration and cellulariza-
tion follow immediately (Ov8). The seven-celled embryo
sac (Fig. 1H) matures with the polarization of the egg
cell and the synergids, and the fusion of the two polar
nuclei in the central cell (Ov9). The egg cell becomes
highly vacuolate at its chalazal pole (Fig. 1I), with the
vacuoles occupying one-third of the entire cell mass.
The cell wall at this pole is distinctly wavy, making the
egg cell easy to distinguish from the synergids. The
polarity of the synergids is opposite to that of the egg
cell.
In the synergids, the vacuoles are positioned at the
micropylar pole. The nucleoli of the diploid central cell
are remarkably larger than that of the egg cell (Fig. 1I).
Initially, three antipodal cells are laterally located at
the chalazal pole of the embryo sac. During embryo sac
maturation, these antipodal cells enlarge and divide
(data not shown) and some become multinucleate.
Division of the antipodals produces six to eight cells.
Nucellus cells lining the sides of the egg apparatus
continue to divide, in either the anticlinal or periclinal
240 LOPEZ-DEE ET AL.
Fig. 1. Light microscopy of the development of the rice ovule. A:
The developing ovule is oriented toward the abaxial side of the pistil.
Ovule development is tenuinucelluste starting at approximately 70°
from the vertical axis and curving downward to about 160°. ⫻20. The
inner (ii) and outer integuments (oi) have been initiated. B: The
archespore (ar) develops from a subepidermal cell of the nucellus. It is
much larger than the other nucellus cells. ⫻100. C: The archespore
has differentiated into the megaspore mother cell (mc). It shows a
characteristically less dense cytoplasm and polarity, with the nucleus
occupying the micropylar half of the cell. ⫻100. D: Linear tetrad of
megaspores. ⫻100. The chalazal megaspore (panel 4) becomes the
functional megaspore. E: Three megaspores at the micropylar end
degenerate (dm) and create space for the functional megaspore (fm) to
divide. ⫻20. The ovule is now about 100° from the vertical axis. F:
Functional megaspore. ⫻100. G: The two-nucleate developing embryo
sac showing vacuolisation at its chalazal end. ⫻40. H: The mature
embryo sac. ⫻10. The antipodals are laterally positioned close to the
chalazal pole. The egg apparatus and central cell occupy the micropy-
lar end. I: The egg apparatus with polarized egg cell (ec) and one
visible synergid (sy) ⫻40. The two polar nuclei of the central cell (cc)
have fused, shown here with two large nucleoli along a strand of
cytoplasm against the egg cell. The micropyle is formed by a narrow
opening in the inner integument, continuous with the intercellular
space between four large nucellus cells lining the egg apparatus.
 MADS-BOX GENES AND RICE DEVELOPMENT 241

Fig. 2. OsMADS13 amino acid sequence. The conserved MADS-box

region is in boldface. The GenBank accession number for the nucleo-
tide sequence is AF151693.

plane. Directly below the egg apparatus, however, a

single large nucellus cells remains.
OsMADS13 Isolation and Sequence Analysis
We isolated OsMADS13 by screening a HybriZap
cDNA library made from RNA of developing ovaries.
This library was screened using mixed probes of differ-
ent MADS boxes of the rice gene OsMADS45 [Greco et
al., 1997], the Petunia gene FBP11 [Angenent et al.,
1995] and the maize ZAG2 gene [Schmidt et al., 1993].
Sequence analysis of OsMADS13 showed that it en-
codes a putative protein of 270 amino acids, containing
the MADS box domain between amino acids 1 and 57 Fig. 3. Dendrogram obtained with the program PHYLIP. Compari-
son of the amino acid sequences of some MADS-box proteins belonging
and the K box domain between the amino acids 93 and
to the AGAMOUS monophyletic group: ZAG2, ZAG1 [Schmidt et al.,
159 (Fig. 2). 1993], ZMM1 and ZMM2 [Theissen et al., 1996] from maize; AGAMOUS
Comparison of the putative translation product of [Yanofsky et al., 1990] and AGL11 [Rounsley et al., 1995] from Arabidop-
OsMADS13 with MADS box proteins from other species sis; FBP7, FBP11, FBP6 [Angenent et al., 1993, 1995], and FBP14
shows that OsMADS13 belongs to the AGAMOUS [Tsushimoto et al., 1993] from Petunia; OsMADS3 [Kang et al., 1995]
from rice; and PLENA [Bradley et al., 1993] from Antirrhinum.
phylogenetic group [Purugganan et al., 1995; Theissen
et al., 1996]. To visualize the homology between the
OsMADS13 amino acid sequence and other members
belonging to the monophyletic AG group [Purugganan,
1995; Theissen et al., 1996], multiple alignment was
performed. This alignment was used to construct a
dendrogram, shown in Figure 3. Within the group of
rice MADS box proteins, OsMADS13 is most homolo-
gous to OsMADS3 (74% similarity, 56.8% identity), the
true rice orthologue of AGAMOUS [Kang et al., 1995].
In general, OsMADS13 is most related to two maize
MADS box proteins, namely ZAG2 and ZMM1, with Fig. 4. RNA blot analysis using OsMADS13 as a probe. Total RNA
which it shares 85.8% and 85.7% similarity, respec- (10 µg) isolated from (1) roots, (2) leaves, (3) inflorescence, (4) ovules,
tively. Furthermore, comparison of the amino acid (5) kernel at 3 days after pollination (3 DAP), (6) 4 DAP, (7) 5 DAP, and
(8) 9 DAP.
sequence of OsMADS13 with the amino acid sequences
of the Petunia ovule specific proteins FBP7 and FBP11
[Angenent et al., 1995] and AGL11 [Rounsley et al., have performed in situ hybridization analysis using an
1995] from Arabidopsis showed greater than 75% simi- OsMADS13 antisense RNA probe. As shown in Figure
larity. 5A,B, OsMADS13 is not expressed during the early
stages of spikelet development. The first detectable
OsMADS13 Is Specifically Expressed OsMADS13 expression is found in ovule primordium
in the Ovule (Fig. 5C,D), where it persists during the further devel-
RNA blot analysis using total RNA from roots, leaves, opment of the ovule (Fig. 5E–H). Inside the ovule,
inflorescence and developing kernels showed that Os- OsMADS13 is expressed in both integuments and nucel-
MADS13 is expressed in inflorescence and kernel tis- lus tissues (Figs. 5G and 5H). In addition, OsMADS13
sues (Fig. 4). To study the tissue specificity and timing mRNA was also detected in the inner cell layer of the
of OsMADS13 expression during floret development we carpel wall (Fig. 5D,F).
242 LOPEZ-DEE ET AL.
Fig. 5. In situ hybridization of developing rice florets. A: No
hybridization signal could be detected in very young spikelets. B:
Developing spikelet at a higher magnification, with no hybridization
signal. C: Spikelets from a 1 cm inflorescence show strong OsMADS13
signals in the ovule primordium. The arrow indicates ovule primor-
dium. D: Closeup of spikelet from 1-cm inflorescence showing localiza-
tion of OsMADS13 transcripts in ovule primordium and in the inner
tissue of the ovary wall (arrowhead) lining the ovule. E: Spikelet on a
Mapping the Genomic Position of OsMADS13
OsMADS13 has been genomically mapped as RFLP
marker in the most dense genetic linkage map of rice
produced to date, consisting of 2,275 markers [Harushima
et al., 1998]. Our gene-specific probe identified a single-
band polymorphism when tested on the parental lines
of the mapping populations Kasalath and Nipponbare
(data not shown). OsMADS13 co-mapped with marker
R3375, located on the short arm of chromosome 12, at
position 39.7. The OsMADS13 map position is sus-
tained by a log-likelihood of ⫺739.78. The same map
position was identified using a probe from the maize
ZAG2 gene, the gene that, together, with its paralogue
ZMM1, shares the highest degree of sequence similar-
ity with OsMADS13. The ZAG2 probe identified a
single band polymorphism and fell into the linkage
group with a log-likelihood of ⫺721.92. It is worth
mentioning that for mapping OsMADS13 and ZAG2,
different restriction enzymes were used for detection of
the polymorphism.
Our cDNA clone ZmOv23 (accession number U31522),
which turned out to be the cDNA of ZMM1 (personal
data), was also used on rice as an RFLP marker. The
same single polymorphic band produced by ZAG2 was
identified, demonstrating that the two maize paralogue
genes identify one single locus on rice chromosome 12.
3-cm inflorescence showing intense signals in the developing ovule.
Arrow indicates ovule primordium. F: OsMADS13 transcripts are
detected in the developing ovule of spikelet from a 5-cm inflorescence.
G: OsMADS13 transcripts are preferentially expressed in the differen-
tiating integuments of the ovule. H: With maturation of the embryo
sac, weaker signals of OsMADS13 transcription are detected in the ovule.
sp, spikelet; le, lemma; ca, carpel primordium; p, palea; ov, ovule
primordia; ow, ovary wall; in integument, es, embryo; sac, nu, nucellus.
DISCUSSION
A molecular genetic approach to the study of rice
ovule development requires detailed knowledge of the
morphological and developmental events that occur
during ovule development. Thus, we have described
rice ovule development in detail in order to provide a
framework for defining the expression patterns of genes
that have putative roles in ovule development, and also
for mutant analysis. We have divided rice ovule develop-
ment into nine distinct morphological stages. This
staging is based on the type of cell(s) formed, the
division processes taking place, and the prominent
cellular structures and positions observed during the
stages. An additional parameter used in this develop-
mental calendar is the formation of the integuments.
The integuments are initiated before megasporogenesis
and develop before the embryo sac is formed (develop-
mental stages Ov2 to Ov4). The curvature of the
developing ovule is also taken into account. The rice
ovule rotates by about 90° from its original position.
Monosporic-Polygonum-type embryo sac develop-
ment is the most common form among flowering plants
[Maheshwari, 1950; Willemse and van Went, 1984;
Reiser and Fischer, 1993]. Rice ovule and embryo sac
development is basically consistent with observations
made of this type of development. Meiosis of the rice
 MADS-BOX GENES AND RICE DEVELOPMENT
megasporocyte results in linear or T-shaped tetrads as
is observed in most grasses. The functional megaspore
in rice forms small vacuoles (Fig. 1F) which are typical
of Polygonum type megagametogenesis [Maheshwari,
1950; Willemse and Van Went, 1984; Haig, 1990; Reiser
and Fischer, 1993]. The rice megagametophyte initially
forms a vacuole at the chalazal pole during the two-
nucleate stage and later forms the large central vacuole
at the late two-nucleate stage, which persists during
the four-nucleate stage. The division of the antipodals,
which occurs after cellular partitioning in the rice
embryo sac, conforms with observations made on the
maize embryo sac. Vollbrecht and Hake [1995] counted
up to almost 100 cells in maize after mitotic division of
the antipodal cells.
Genomic mapping is a fundamental aspect in the
characterization of novel genes. In this respect, the
availability of highly dense genetic maps that cover the
entire genome are a great advantage. This situation
can be usefully exploited in rice, for which extensive
genetic and physical mapping with various types of
molecular markers have produced a large amount of
information regarding genome structure, gene position,
and the identification of quantitative trait loci control-
ling agronomically important characters. The impor-
tance of gene mapping in rice is also enhanced by the
extensive colinearity found among the genomes of
grasses for which rice, with its compact genome, repre-
sents the species of reference [Moore et al., 1995, 1997].
In our case, RFLP mapping proved very useful in
defining the phylogenetic relationships among MADS
box genes in maize and rice, for which sequence compari-
son and analogy of expression patterns have suggested
a common ancestor and functional similarity.
OsMADS13 shares the highest degree of homology
with the cDNA clones of ZAG2 [Schmidt et al., 1993]
and ZMM1 [Theissen et al., 1995]. ZAG2 and ZMM1 are
located in duplicated regions of the maize genome, on
chromosomes 3 and 10, respectively. Mapping within
and across species of the grass family has produced a
consensus grass comparative map and has substanti-
ated the hypothesis that maize is allotetraploid [Devos
and Gale, 1997]. According to this hypothesis, the
duplicated regions comprising the two paralogous genes
ZAG2 and ZMM1 derive from allotetraploidation. Our
mapping data clearly indicate that OsMADS13 is the
ortholog of the ZAG2/ZMM1 pair. OsMADS13 maps as
a single locus on the short arm of rice chromosome 12
which, according to the consensus grass comparative
map is considered to be colinear with both maize
chromosomes 3 and 10, the linkage group locations of
ZAG2 and ZMM1, respectively. However, this consen-
sus map can be viewed only as indicative, unless direct
cross mapping of the genes of interest is performed. Our
cross-mapping data, in fact, confirm the syntenic rela-
tionships between maize chromosomes 3 and 10 and
rice chromosome 12 and add strong evidence support-
ing the hypothesis that OsMADS13 is phylogenetically
related to ZAG2 and ZMM1.
243
In situ hybridization analysis showed that Os-
MADS13 expression was detectable only in the later
stages of flower development, when the ovule starts to
develop. Its expression was restricted to developing
ovules and the inner site of the carpel wall and per-
sisted until the ovule was mature. Comparison of the in
situ data with the histological analyses (Fig. 1) showed
that OsMADS13 expression was largely restricted to
the sporophytic parts of the ovule.
Comparative sequence analysis (Fig. 3) shows that
OsMADS13 shares high homology with OsMADS3,
which is considered the rice homologue of AGAMOUS
from Arabidopsis [Kang et al., 1995; Yanofsky et al.,
1990]. Comparing the in situ hybridization data shows
that these genes are expressed differently. OsMADS3
is, like AGAMOUS in Arabidopsis, expressed both in
stamens and carpel, whereas OsMADS13 is only ex-
pressed in the inner part of the carpel. The different
expression patterns of these two rice MADS-box genes
suggests that they will not have a comparable function
in flower development.
The expression pattern of OsMADS13 is very similar
to that observed for ZAG2 in maize, which is also
expressed in developing ovules and in the inner carpel
faces [Schmidt et al., 1993]. For both monocot homologs,
OsMADS13 and ZAG2, no function has yet been deter-
mined, although their patterns of expression suggest
that they may play important roles in ovule develop-
ment. Experiments are currently under way to overex-
press and suppress OsMADS13 in rice, which may help
us to understand the function of this gene. For two
genes of Petunia, FBP7 and FBP11, these experiments
have already been done. Experiments in which FBP7
and FBP11 expression was suppressed showed that
these genes are necessary to determine ovule develop-
ment. Their overexpression resulted in ectopic ovule
formation on sepals and petals, indicating that they are
sufficient to induce ovule formation. FBP7 and FBP11
are most likely orthologues of OsMADS13 because they
exhibit similar expression patterns and show high
levels of homology (greater than 75%). Given these
observations, one might speculate that OsMADS13 and
ZAG2 will have a comparable role in rice and maize
ovule development, respectively.
ACKNOWLEDGMENTS
We thank Dr. Andrè Van Lammeren for helpful
comments, and Dr. Andrew MacCabe for critical read-
ing of the manuscript. We thank Dr. T. Sasaki, Dr. M.
Yano, and Dr. A. Shomura of the Rice Genome Project at
Tsukuba, for providing assistance for the mapping
experiments. Furthermore, we thank Dr. Simona Masi-
ero and Dr. Diana Rigola for their assistance in some of
the experiments. Short working visits by Zenaida Lopez-
Dee and Dr. Peter Wittich to Holland and Italy, respec-
tively, were supported by the collaborative NATO grant
CRG.CRG 973222.
244 LOPEZ-DEE ET AL.
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