The Plant Cell, Vol. 13, 481–493, March 2001, www.plantcell.org © 2001 American Society of Plant Physiologists
Sex Determination in the Monoecious Species Cucumber Is
Confined to Specific Floral Whorls
Martin M. Kater,1 John Franken, Kim J. Carney,2 Lucia Colombo,3 and Gerco C. Angenent 4
Plant Research International, Business Unit Plant Development and Reproduction, 6700 AA Wageningen, The Netherlands
In unisexual flowers, sex is determined by the selective repression of growth or the abortion of either male or female
reproductive organs. The mechanism by which this process is controlled in plants is still poorly understood. Because it
is known that the identity of reproductive organs in plants is controlled by homeotic genes belonging to the MADS box
gene family, we analyzed floral homeotic mutants from cucumber, a species that bears both male and female flowers
on the same individual. To study the characteristics of sex determination in more detail, we produced mutants similar
to class A and C homeotic mutants from well-characterized hermaphrodite species such as Arabidopsis by ectopically
expressing and suppressing the cucumber gene CUCUMBER MADS1 (CUM1). The cucumber mutant green petals (gp)
corresponds to the previously characterized B mutants from several species and appeared to be caused by a deletion
of 15 amino acid residues in the coding region of the class B MADS box gene CUM26. These homeotic mutants reveal
two important concepts that govern sex determination in cucumber. First, the arrest of either male or female organ development is dependent on their positions in the flower and is not associated with their sexual identity. Second, the
data presented here strongly suggest that the class C homeotic function is required for the position-dependent arrest
of reproductive organs.
INTRODUCTION
In flowering plants, strategies for sex determination have
evolved to prevent self-fertilization that may lead to loss of
fitness due to inbreeding (Darwin, 1876). One of these
strategies involves the production of unisexual flowers, in
which male and female gametes are segregated on different flowers of the same plant (monoecious species) or on
separate individuals (dioecious species). Differences occur
in the manner in which the reproductive organs are arrested; in maize, for example, the sex organ primordia
abort completely (Dellaporta and Calderon-Urrea, 1994),
whereas in cucumber, these primordia arrest in their
growth (Malepszy and Niemirowcz-Szcytt, 1991; Grant et
al., 1994).
Although sex determination in animals is well studied, little is known about the molecular control of this process in
plants. To date, only two genes have been cloned that influ-
1 Current
address: Dipartimento di Genetica e Biologia dei Microrganismi, Università di Milano, via Celoria 26, 20133 Milan, Italy.
2 Current address: Seminis Vegetable Seeds, Inc., 37437 State Highway 16, Woodland, CA 95695.
3 Current address: Dipartimento di Biologia, Università di Milano, Via
Celoria 26, 20133 Milan, Italy.
4 To whom correspondence should be addressed. E-mail g.c.angenent
@plant.wag-ur.nl; fax 31-317-418094.
ence sex in maize. The TASSELSEED2 (TS2) gene, which
encodes a predicted protein with significant homology with
a steroid-specific dehydrogenase from bacteria, is involved
in the abortion of pistil primordia in the tassel (DeLong et al.,
1993). In contrast, the feminizing ANTHER EAR1 (AN1) gene
(Bensen et al., 1995), which encodes an enzyme in the gibberellin biosynthesis pathway, is required for stamen abortion in the maize ear.
The basic architecture of well-studied hermaphrodite
flowers such as those in Arabidopsis, snapdragon, and petunia consists of four concentric whorls with, starting from
the outside, the sepals (whorl 1), petals (whorl 2), stamens
(whorl 3), and finally the carpels (whorl 4). The identity of the
floral organs is specified by three distinct classes of floral
homeotic genes (A, B, and C). The combinatorial action of
these ABC genes has been described in a model (reviewed
in Coen and Meyerowitz, 1991) that is based on studies of
homeotic flower mutants from Arabidopsis and snapdragon
(Carpenter and Coen, 1990; Schwarz-Sommer et al., 1990;
Bowman et al., 1991). In whorl 1, the A genes control the
development of the sepals; in whorl 2, both class A and B
genes are active, leading to the formation of petals. In whorl
3, the formation of stamens is defined by the combinatorial
action of class B and C homeotic genes; the identity of the
primordia of whorl 4 is determined by the class C genes
only. In addition, the A and C gene functions are mutually
antagonistic.
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The genes belonging to the B and C classes are members
of the MADS box gene family. These genes encode putative
transcription factors with a highly conserved DNA binding
domain called the MADS box. Besides the MADS box, these
genes encode a second, more moderately conserved domain called the K box, which is involved in dimerization of
the transcription factors (Davies et al., 1996; Fan et al.,
1997). MADS box genes have been isolated from dioecious
species such as white campion (Hardenack et al., 1994) and
sorrel (Ainsworth et al., 1995) and from monoecious species
such as maize (Schmidt et al., 1993; Mena et al., 1995) and
cucumber (Kater et al., 1998; Perl-Treves et al., 1998). In
male and female flowers of white campion and sorrel, the
expression patterns of several MADS box genes were analyzed to understand the role of MADS box genes in sex determination. In white campion, the expression patterns of
the two proposed class B genes, SLM2 and SLM3, are influenced by the sex of the plant. In male flowers, the transcripts were detected closer to the center of the floral
meristem than in female flowers, and this finding was correlated with a reduction in the size of whorl 4 (Hardenack et
al., 1994). In sorrel flowers, inappropriate primordia arrest
very early in their development. It was observed that the
proposed class C gene RAP1 was expressed very transiently in whorls 3 and 4 of male and female flowers, but its
expression became undetectable in the arrested primordia
(Ainsworth et al., 1995). Although these studies show that
the expression patterns of the organ identity MADS box
genes depend on the sexual identity of the flower, the question of whether these genes play an active role in the sex
determination process remains.
Here, we studied homeotic floral mutants from the monoecious species cucumber that were generated by modifying
the expression of MADS box transcription factor genes belonging to classes B and C. This unique set of mutants provides novel information about the spatial control of sex
determination and suggests a role of the class C homeotic
genes in controlling the arrest of the reproductive organs in
cucumber.
RESULTS
Macroscopic and Microscopic Analyses of the
Cucumber Flower
Male cucumber flowers are composed of four whorls of organs (from the outer to the inner whorl): five sepals, five
yellow petals, five stamens, and three arrested carpel primordia in the fourth whorl (Figures 1A to 1C). In contrast,
stamens are arrested in their development in the female
flower, and the three carpel primordia develop further to an
inferior ovary, a short style, and three separated stigmas
(Figures 1D to 1F). To identify stamen primordia in female
flowers, we made histological sections of young flower
buds. These sections show arrested stamens in the third floral whorl (Figures 1D and 1E).
Isolation and Expression Analysis of Cucumber Class B
and C MADS Box Genes
Due to the high level of sequence conservation within the
MADS box domain, MADS box genes have been isolated
from various species by using heterologous probes. In this
study, class B and C MADS box genes were isolated from
cucumber by screening a cucumber female flower–specific
cDNA library by using petunia MADS box genes as a probe.
One of them, designated CUCUMBER MADS1 (CUM1;
Kater et al., 1998), is very similar in its deduced amino acid
sequence and expression pattern to the class C genes
AGAMOUS (AG) from Arabidopsis (Yanofsky et al., 1990)
and PLENA (PLE) from snapdragon (Bradley et al., 1993).
Another MADS box gene that was isolated, designated
CUM26 (GenBank accession number AF043255), is most likely
the ortholog of the previously characterized class B genes PISTILLATA (PI) from Arabidopsis (Goto and Meyerowitz, 1994),
GLOBOSA (GLO) from snapdragon (Schwarz-Sommer et
al., 1992), and FLORAL BINDING PROTEIN 1 (FBP1) from
petunia (Angenent et al., 1992). The deduced protein sequence of CUM26 (Figure 2A) shows that it has 69, 70, and
71% of its amino acid residues in common with PI, GLO,
and FBP1, respectively.
The expression patterns of CUM1 and CUM26 were initially studied by RNA gel blot analysis using RNA extracted
from leaves and the various floral organs (Figure 2B). To
avoid cross-hybridization due to sequence homology between MADS box genes, we hybridized the RNA gel blot
with probes derived from the divergent 3⬘ ends of the cDNAs.
This experiment showed that CUM1 was expressed in stamens and throughout the pistil in the style, stigma, nectary,
and ovary. CUM26 expression was similar to that of other
class B genes, restricted to the second and third floral
whorls.
The expression profiles of these two MADS box genes
were examined in more detail by in situ hybridizations on
longitudinal sections of young male and female cucumber
flower buds. Figure 3A shows hybridizing signals in stamens
and arrested pistil primordia from young male flowers by using antisense RNA derived from the class C gene CUM1 as
a probe. In female flowers, CUM1 mRNA accumulated at a
low level in stamen primordia that were arrested in development. In pistils, CUM1 was expressed in the stigma, the
ovules, and the placenta, whereas no expression was observed in other parts of the ovary (Figure 3B). CUM26 was
expressed in male flowers in petals and young stamens,
which is to be expected from a class B gene (Figure 3C).
Similarly, in a female flower, CUM26 transcripts were detectable in petals and arrested stamen primordia (Figure
3D). These data demonstrate that the presumed class B and
Sex Determination in Cucumber
483
Figure 1. Flower Morphology of Wild-Type Cucumber Plants.
(A) Longitudinal section through male flower buds at two developmental stages. The carpel primordia are arrested in whorl 4. Stamen primordia
arise from the flanks of the petals and produce sporogenous tissue.
(B) Longitudinal section through a young wild-type male cucumber flower at a later developmental stage just before opening of the flower. The
anthers start to produce pollen.
(C) Macroscopic view of a male flower. A pair of sepals and petals were removed to allow a view inside. The arrested carpel primordia are visible
at the bottom of the flower.
(D) Longitudinal section through a female flower bud. Stamen and carpel primordia develop in whorls 3 and 4, respectively. The sepals cover the
flower completely.
(E) Longitudinal section through a female flower bud at a later developmental stage. The stamen primordia are arrested, and in the fourth whorl
an inferior ovary and superior stigmas develop.
(F) Macroscopic view of a female flower at a stage just before opening of the flower. As in (C), a pair of sepals and petals have been removed to
allow a view inside the flower.
The whorl numbers indicate the positions of the floral organs within the flower. O, ovary. Bars in (A), (B), (D), and (E) ⫽ 1 mm.
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C genes are still expressed in primordia that are arrested in
development.
Phenotype and Molecular Analyses of a Class B
Homeotic Mutant
We studied the homeotic transformations in a spontaneous
recessive cucumber mutant designated green petals (gp).
Young male flowers of this gp mutant consisted of two perianth whorls of sepals, and outgrowth of the reproductive organs was arrested (Figure 4A). The male flower became
indeterminate when the flower subsequently aged, resulting
in a repetition of sepal whorls and a bushy appearance (Figure 4B). Histological analysis of these male gp flowers revealed that the indeterminate flower buds developed from
the third whorl, whereas the fourth inner whorl was arrested
in development (Figures 4C and 4D). Interestingly, when the
mutant was grown at high temperature ( ⬎30⬚C), the mor-
Figure 2. Amino Acid Sequence of CUM26 in the Wild Type and the
gp Mutant and Expression Patterns of CUM1 and CUM26.
(A) The CUM26 protein sequence deduced from the longest reading
frame of CUM26 cDNA. The conserved MADS box is underlined
with a thick line, and the K box region is underlined with a thin line.
The 15 amino acid residues that are deleted in the CUM26 protein of
the gp mutant are boxed.
(B) CUM1 and CUM26 expression in cucumber leaves and floral organs. Total RNA was isolated from mature leaves (L), sepals (S), petals (P), stamens (St), styles (Sl), stigmas (Sg), nectaries (N), and
inferior ovaries (O).
phology of the male flower changed dramatically (Figures 4E
and 4F): sepals were still formed in the first two outer
whorls, but its indeterminate character was lost, and carpels
instead of stamens were produced in the third whorl. In an
older male flower (Figure 4F), the whorl 3 carpels developed
into complete parthenocarpic fruit that were positioned superior to the receptacle. This mutant phenotype shows
clearly that female organs can develop in male flowers.
Analysis of longitudinal sections of these male flowers at
different stages of development indicated that the whorl 4
primordia were also arrested in growth at high temperature.
Furthermore, these microscopic analyses confirmed that the
superior carpels originated from the third whorl, whereas
whorl 4 primordia were still arrested in development (Figures
4H and 4I). The homeotic conversions observed at high
temperature resembled exactly those of class B mutants in
species with bisexual flowers, such as Arabidopsis, snapdragon, and petunia (Coen and Meyerowitz, 1991; Angenent
et al., 1993). The female flowers, which can be easily recognized by their inferior ovary, produced sepals in both outer
whorls, and no obvious differences in whorl 3 and 4 organs
were observed between flowers from the gp mutant and
wild-type plants (Figures 4G and 4J). In contrast with the
male flowers, the female flowers were not sensitive to different temperature conditions.
To investigate the molecular nature of this gp mutant, we
cloned the CUM26 coding sequence by using reverse transcription–polymerase chain reaction (PCR) on RNA from
very young gp flower buds. Sequence analysis demonstrated that this gene contains an in-frame deletion of 15
amino acids just downstream from the region encoding the
K domain (Figure 2A), a motif shown to be involved in protein–protein interactions between MADS box proteins (Davies
and Schwarz-Sommer, 1994; Davies et al., 1996; Fan et al.,
1997). For DEFICIENS (DEF), a class B gene of snapdragon,
and AG, the class C gene of Arabidopsis, it has been shown
that mutations in the K domain lead to temperature-sensitive phenotypes (Sieburth et al., 1995; Zachgo et al., 1995).
Apparently, the deletion in CUM26 affects the function of
the protein at high temperatures, resulting in the gp mutant
phenotype.
The gp mutant phenotype is linked to this deletion in the
CUM26 protein, as demonstrated in a population segregating for the mutant and wild-type phenotypes. In all mutants
analyzed (eight plants), the deletion was confirmed by PCR
analysis, whereas in the plants with the wild-type phenotype
(18 plants), either only the wild-type or both the wild-type
and mutant alleles were present (data not shown).
Expression Analysis of CUM1 and CUM26 in gp
Mutant Flowers
To analyze the expression of class B and C MADS box
genes in male gp flowers in detail, we performed in situ hybridization analysis. Figures 3E and 3F show that CUM26
Sex Determination in Cucumber
485
Figure 3. Expression Patterns of CUM1 and CUM26 in Wild-Type and gp Mutant Flowers.
Longitudinal sections were hybridized with digoxigenin-labeled antisense CUM1 ([A], [B], [G], and [H]) or CUM26 ([C], [D], [E], and [F]) RNA. All
sections were viewed using dark-field microscopy. The whorl numbers indicate the positions of the floral organs within the flower.
(A) and (C) Young male flower buds from a wild-type plant. The carpel primordia are arrested and the anthers start to produce sporogenous tissue. The bud is completely covered by sepals.
(B) and (D) Young female flower buds from a wild-type plant. The stamen primordia are arrested and in the fourth whorl an inferior ovary and superior stigmas develop.
(E) and (G) Male flowers from a gp plant grown under normal temperature conditions (22⬚C). The red arrowheads indicate new buds as they appear in these bushy indeterminate flowers (cf. Figure 4B).
(F) and (H) Male flowers from a gp plant grown under high-temperature conditions (35⬚C). Carpels are formed in whorl 3, and the carpel primordia in the fourth whorl are arrested. The outer two whorls are sepals.
O, ovary. Bar in (A) ⫽ 1 mm for (A) through (H).
transcripts were not detectable under normal (22 ⬚C) and
high-temperature (35⬚C) conditions in male and female (not
shown) gp flowers, indicating that the class B function necessary for normal petal and stamen development was not
present in this mutant. Surprisingly, hybridization with a
CUM1 antisense probe did not reveal any signal in a gp
male flower grown at 22⬚C when no reproductive organs developed (Figure 3G). In contrast, the same probe detected
CUM1 transcripts in the third and fourth floral whorl primordia of plants grown at high temperature (Figure 3H). Due to the
absence of class B gene expression (CUM26) and the expression of CUM1, whorl 3 primordia have a carpel identity ac-
cording to the ABC model. This was confirmed at later stages
of development by the visible outgrowth of superior carpels in
the third whorls of these flowers (Figures 4E and 4F).
Ectopic Expression of CUM1 in Cucumber Induces
Reproductive Organ Formation in the First and Second
Floral Whorls
It has been demonstrated that Arabidopsis plants overexpressing AG under the control of the cauliflower mosaic virus (CaMV) 35S promoter phenocopy apetala2 mutant
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The Plant Cell
Figure 4. Flower Morphology of the gp Mutant.
Sex Determination in Cucumber
plants, confirming the model’s prediction that class A suppresses the class C function in the two outer whorls
(Mizukami and Ma, 1992). Overexpression of CUM1 driven
by the CaMV 35S promoter in the hermaphrodite species
petunia resulted in similarly severe homeotic transformations of sepals into carpelloid structures and petals into stamens (Kater et al., 1998). However, in the monoecious
species cucumber, reproductive organs were arrested in the
two types of flowers, raising the question of whether this
phenomenon also occurs when the reproductive organs develop at other positions in the flower. Therefore, the CUM1
overexpression construct initially used for the petunia transformations was introduced in cucumber. Two independent
transformants, T340-1 and T340-5, showed the most severe
and identical homeotic transformations, and in both plants,
the CUM1 gene was expressed ectopically, as confirmed by
RNA gel blot analysis (data not shown). Although the severe
homeotic transformations affect the floral structure significantly, male and female flowers were easily distinguished,
because the female flowers still developed an inferior ovary
that is not present in male flowers. In whorl 1, the sepals of
both male and female flowers of these lines were transformed into carpelloid structures with stigmatic tissue on
top (Figures 5A to 5E). Petals were reduced in size significantly or completely absent in both male and female flowers. Histological analysis was performed to determine the
identities of the chimeric organs in whorl 2. This analysis revealed that antheroid tissues, including pollen grains, develop on top of the whorl 2 organs in both male and female
flowers (Figures 5B, 5C, 5E, and 5F). As shown in Figure 5D,
the ovaries of these female flowers were malformed, which
might be a secondary effect of the aberrations in the other
floral organs. The floral phenotypes of these transgenic
plants demonstrate that in the second whorl of female flowers male tissue is allowed to develop, whereas in wild-type
flowers male organ formation never occurs. Furthermore, female organs develop in the first whorl of male mutant flow-
487
ers, converting it from a unisexual to a bisexual cucumber
flower.
Downregulation of CUM1 Results in a Class C
Mutant Phenotype
In addition to the plants ectopically expressing CUM1, the
same transformation experiment revealed three cosuppression plants in which endogenous CUM1 expression was
abolished completely, as determined by RNA gel blot hybridization (data not shown). Figures 5G to 5L show that the
cosuppression phenotypes are similar but distinct from that
of the Arabidopsis ag mutant. The loss of AG function in Arabidopsis resulted in homeotic transformations of stamens
into petals, and a reiteration of the floral program in the center of these flowers replaced the pistil (Figure 6; Pruitt et al.,
1987). Surprisingly, in the male cucumber flowers of the cosuppression plants, five new floral buds appeared in the
third whorl (Figures 5G and 5H). These new flower buds
were arranged as two couples and a separate bud occupying the same positions as stamens in wild-type male flowers. In the center of the mutant flower, the rudimentary
carpel primordia were replaced by a new indeterminate
flower similar to that seen in the Arabidopsis ag mutant
flower.
In female flowers in which CUM1 was cosuppressed, petals developed in the third whorl and the floral program reiterated in the center of the flower, with a small fruit growing
inside the primary ovary (Figure 5K, arrow). A cross-view
through a female floral bud demonstrated that the whorl 3
primordia were not arrested and developed into petalloid organs (Figure 5L). The observation that ovaries still develop in
these female flowers suggests that CUM1 expression is not
required or redundant for the development of the fruit, which
is in agreement with the absence of CUM1 expression in the
fruit of wild-type flowers (Figure 3B).
Figure 4. (continued).
(A) Young male flower of the gp mutant grown at 22⬚C. The flower is composed of two whorls of sepals only.
(B) Older indeterminate male flower grown at 22⬚C. New buds develop inside the primary flower, which results in a bushy appearance.
(C) Longitudinal section through young male flower buds grown at 22⬚C. Initially, the whorl 3 and 4 primordia develop as in the wild type (left
bud). At a later developmental stage (right bud), new meristems develop in the third whorl, as indicated by the red arrowhead. The carpel primordia in whorl 4 remain arrested.
(D) Longitudinal section through an older indeterminate male flower grown at 22⬚C. A new flower bud originating from the third whorl in this
bushy flower is indicated by the red arrowhead.
(E) Male flower of the gp mutant grown under high-temperature conditions (35⬚C) with stamens homeotically transformed into carpels.
(F) Older male flower of the gp mutant grown under high-temperature conditions (35⬚C) with fruit developing in whorl 3. The whorl 1 and 2 organs are senesced.
(G) Female flower of the gp mutant grown at 22⬚C. The inferior ovary is not affected, and the outer two whorl organs are sepals. No changes in
flower phenotype were observed when the plants were grown under high-temperature conditions (35⬚C).
(H) Longitudinal section through a young male flower of the gp mutant grown under high-temperature conditions (35⬚C). Carpelloid structures
develop in whorl 3, and carpel primordia are arrested in whorl 4.
(I) Older bud as in (H). Fruit-like bodies develop in the third whorl on positions normally occupied by stamens (cf. Figures 4E and 4F).
(J) Longitudinal section through a female flower bud of the gp mutant. The two inner whorls are like those in wild-type flowers (cf. Figure 1E).
The whorl numbers indicate the positions of the floral organs within the flower. O, ovary. Bars in (C), (D), (H), (I), and (J) ⫽ 1 mm.
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Figure 5. Flower Morphology of Transgenic Cucumber Plants in Which CUM1 Was Ectopically Expressed or Cosuppressed.
Sex Determination in Cucumber
Interestingly, in these cosuppression flowers, nonreproductive organs were allowed to form in the two inner whorls
of male and female flowers at positions where the development of reproductive organs was normally arrested.
DISCUSSION
To gain insight into the process of sex determination, we analyzed homeotic floral mutants of the monoecious species
cucumber in which female flowers produce male organs and
vice versa. Three types of mutants similar to the known
class A, B, and C mutants from hermaphrodite flowers (Figure 6) were analyzed. Class A and C mutants were obtained
by transforming cucumber with a construct containing the
cucumber class C MADS box gene CUM1 under the control
of the CaMV 35S promoter, which resulted in ectopic expression and cosuppression of this gene, respectively. The
class B mutant, gp, was a spontaneous recessive mutant already present in our collection. Obviously, this study does
not provide a complete picture of how sex determination is
controlled, because phytohormones also play an important
role in the determination of sex, as has been demonstrated
in many monoecious and dioecious species by the application of hormones. In addition, mutants in maize, in particular
the anther ear and dwarf mutants, suggest the role of hormones in sex determination (Phinney, 1956; Dellaporta and
Calderon-Urrea, 1994; Bensen et al., 1995). The data reported here reveal novel information regarding the role of
floral organ position and identity in the control of sex determination.
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Temperature-Sensitive Class B Mutant
The phenotype of the female flower in the class B mutant
mimicked that of class B mutants (Figure 6) of hermaphrodite flowers except for the whorl 3 organs. The cucumber
class B mutant had sepals in the first two whorls and a carpel in the fourth whorl. The third whorl carpel did not develop in these female flowers, despite the expected female
identity of this organ. Also, the male flowers of this gp mutant had a phenotype that could be predicted by the ABC
model (sepals-sepals-carpels–arrested primordia); however,
this phenotype was obtained only when these plants were
grown at high temperature (⬎30⬚C). Under conditions of
moderate temperature, the male gp flowers consisted only
of sepals, which became indeterminate in flowers that arose
later. Longitudinal sections showed clearly that the indeterminate flower buds arose from the third floral whorl in which
stamens normally developed. This finding is in contrast with
observations in hermaphrodite flowers, in which only the
loss of class C function resulted in indeterminacy of the
flower, and only in the fourth whorl (Bowman et al., 1989;
Schwarz-Sommer et al., 1990). We hypothesize that indeterminacy is caused by a loss of class C activity in cucumber
flowers as well, although not only in the fourth whorl but also
in the third floral whorl. This notion is supported by the observation that in male flowers of cucumber plants in which
CUM1 was cosuppressed, indeterminacy was observed in
both inner whorls.
Considering the class C function as the key regulator of
floral meristem determinacy, as in hermaphrodite flowers,
we propose the following explanation for the phenotype of
male gp flowers. Under moderate temperature conditions,
Figure 5. (continued).
(A) Male flower of a CUM1 ectopically expressing plant (T340-1). Five complete superior carpels develop in whorl 1, which are partly fused at the
basis forming an ovary-like structure.
(B) Longitudinal section through a male flower of T340-1. The whorl 1 organs are carpelloid, and the organs in the second whorl are chimeric
with petaloid and antheroid tissue. The carpel primordia in whorl 4 are arrested.
(C) Detail of (B) as indicated by the box. Antheroid tissue developing on top of the second whorl organs. Sporogenous tissue with developing
pollen is indicated by the arrow.
(D) Female flower of T340-1 with superior carpels in whorl 1 and a malformed ovary in the fourth whorl. In the second whorl, remnants of petal
tissue is indicated by an arrow.
(E) Longitudinal section through a female flower of T340-1. Antheroid tissue developing on top of the second whorl organs is indicated in the
box. The inner part of the flower is highly malformed and organs are not recognizable.
(F) Detail of (E) as indicated by the box. An arrow shows the developing pollen in the antheroid tissue.
(G) Longitudinal view of a male flower bud of transformant T340-3 in which CUM1 was cosuppressed. Indeterminate floral buds are visible in
whorls 3 and 4.
(H) Male flower of transformant T340-3 showing indeterminate floral bud formation in positions normally occupied by stamens. The flower is indeterminate in the center.
(I) Female flower of T340-3, with petal formation in the third whorl. The inferior ovary is not affected in this homeotic mutant.
(J) Detail of (I) showing the petals in whorl 3 and the indeterminacy in the fourth whorl.
(K) Longitudinal view of a female flower bud of T340-3. The small fruit growing inside the primary ovary is indicated by an arrow. The line indicates the plane of the cross-section shown in (L).
(L) Cross-section through a female flower bud of T340-3. The position of the section is indicated in (H). Petals develop in whorl 3 on positions
normally occupied by stamens. The whorl 4 structure is the upper part of the small fruit that is growing inside the primary ovary.
The whorl numbers indicate the positions of the floral organs within the flower. S, stigmatic tissue; O, ovary. Bars in (B), (E), and (L) ⫽ 1 mm.
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class C activity is probably low or absent in the third whorl
of the gp flower, which was confirmed by the in situ hybridization experiment (Figure 3G). This, in combination with the
absence of class B gene expression, results in the formation
of flowers that are indeterminate in whorl 3 and composed
of only sepals. The fact that these flowers contain only sepals is in agreement with the ABC model, because class A
expression alone results in sepal formation, as shown for
the Arabidopsis class B and C double mutant ap3 ag, which
is indeterminate in whorl 4 and is composed of only sepals
(Bowman et al., 1989).
Under high-temperature conditions (⬎30⬚C), class C activity apparently increases in the third whorl, leading to the
determination of carpel identity and the loss of indeterminacy. This idea is supported by the in situ hybridization experiments, which clearly showed that CUM1 was expressed
in the third whorl organs of male gp flowers grown at high
temperature (Figure 3H). Surprisingly, we did not find indeterminacy in the third whorl of female gp flowers, even in the
cosuppression plant in which CUM1 expression was inhibited. This suggests that in female flowers determinacy is apparent only in the fourth whorl, as in bisexual flowers.
A possible explanation for the discrepancy in indeterminacy in the third whorl between male and female flowers
might be that male flowers require a second stop function in
the third whorl, because the pistil that normally determines
the flower in the fourth whorl does not develop.
Sex Determination
Figure 6. Scheme of the ABC Model of Floral Organ Identity Determination (Coen and Meyerowitz, 1991; Weigel and Meyerowitz,
1994).
(A) The identity of the floral organs is defined by the actions of three
distinct classes of floral homeotic genes, each of which is active in
two adjacent whorls in wild-type flowers. In the first whorl, only class
A genes are active and lead to the formation of sepals. In whorl 2,
the formation of petals is defined by the combinatorial action of
class A and B genes. The combinatorial expression of class B and C
homeotic genes in whorl 3 determines stamen identity. In whorl 4, in
which only the class C function is active, carpels develop. In addition, the class A and C functions are mutually antagonistic, as indicated by the two-headed arrow.
(B) Ectopic expression of a class C gene leads to the suppression of
the class A function, resulting in a phenotype similar to that of the
class A mutant: homeotic transformations of sepals into carpels and
petals into stamens.
(C) In the class B mutant, only class A and C genes are active, resulting in sepals in the two outer floral whorls and carpels in the two
inner whorls.
(D) In the class C mutant, the class A function is active in all four floral whorls, resulting in petals in whorl 3. Class C genes are also essential to specify meristem determinacy in the most inner whorl.
Loss of class C function therefore results in the development of indeterminate flowers in the fourth floral whorl.
The main goal of this study was to investigate sex determination by using homeotic mutants of a monoecious species.
These mutants will provide clues to how the identity of a floral organ is related to the arrest of reproductive organs,
which is the final effect of the sex determination machinery
in cucumber. Additional information has been obtained on
the role of homeotic genes in this process, which has been
examined previously in studies of white campion (Hardenack
et al., 1994) and sorrel (Ainsworth et al., 1995).
Transformants ectopically expressing CUM1 developed
antheroid tissue with pollen grains in the second whorl organs, which clearly shows the male identity of these organs.
This finding demonstrates that in the second whorl of female
flowers, male tissue is allowed to develop, whereas in wildtype flowers, male organ formation never occurs in any of
the floral whorls. Furthermore, female organs developed in
the first whorl of male and female flowers of this mutant
type, converting male flowers from a unisexual to a bisexual
flower. This result demonstrates that female organ formation
is not arrested in the first whorl of male flowers.
The gp mutant clearly shows that female organ formation
also is allowed in male flowers at a position where normally
stamens are formed. These female organs were never produced in the fourth whorl of these flowers. In the female
flowers, whorl 3 organs were arrested despite their pre-
Sex Determination in Cucumber
sumed female identity. These results provide evidence that
sex determination in male flowers is restricted to the fourth
whorl, whereas in other whorls the sex determination mechanism is not active. Similarly, the female mutant flowers
demonstrate that sex determination is determined by the
position of the restricted organs rather than by its identity.
Based on these results as summarized in Figure 7, we
conclude that the process of preventing carpel development
in male flowers is restricted to whorl 4, whereas the sex determination machinery in female flowers seems to suppress
stamen formation only in the third whorl.
However, there is an exception to this proposed rule. The
outgrowth of nonreproductive organs is not prevented in
these restricted floral whorls, as was observed in the flowers
of transgenic plants in which CUM1 was cosuppressed.
This finding suggests that the sex determination machinery
is able to sense the reproductive identity of the developing
organs in whorls 3 and 4. This reproductive identity is determined by the class C function in the fourth whorl and by the
combination of class B and C functions in the third whorl. It
is tempting to speculate that these homeotic genes are the
sensor targets for the sex determination machinery, although class B and C gene expression is not affected in the
arrested organs. The fact that some flowering plants have
evolved a mechanism that selectively prevents the outgrowth of reproductive organs without affecting the development of nonreproductive organs is in agreement with the
presumed evolutionary function of sex determination as a
strategy to control cross-pollination.
491
METHODS
Screening of a cDNA Library
A young female cucumber (Cucumis sativus) flower–specific cDNA library was screened with petunia class B and C MADS box cDNAs,
as described recently by Kater et al. (1998). cDNAs encoding CUM1
were identified using 3⬘ gene-specific cDNA fragments of the petunia
class C cDNAs encoding pMADS3 (Tsuchimoto et al., 1993) and Floral Binding Protein 6 (FBP6; Angenent et al., 1993) as probes. cDNAs
encoding CUM26 were identified using the 3⬘ gene-specific part of
the class B gene FBP1 (Angenent et al., 1992).
Construction of the Binary Vector and Plant Transformation
The cloning of CUM1 cDNA under the control of the double cauliflower
mosaic virus (CaMV) 35S promoter was described recently by Kater et
al. (1998). Leaf tissue from one proprietary Asgrow Seed Company
(Woodland, CA) inbred beta alpha cucumber line was transformed using the leaf transformation procedure described by Horsch et al. (1985)
and Asgrow’s proprietary media formulations, which are conducive to
regenerating cucumber from leaf tissue. Calli were initiated and shoots
were regenerated and rooted in the presence of kanamycin. Twenty
independent transgenic plants were obtained.
Microscopy
For light microscopic analysis, the material was fixed, sectioned, and
stained according to Angenent et al. (1995).
RNA Gel Blot Analysis and In Situ Hybridization
Total RNA was isolated from cucumber leaves or mature floral tissues according to Verwoerd et al. (1989). Subsequently, 10 ␮g of glyoxal (1.5 M) denatured total RNA was separated by electrophoresis
and blotted onto Hybond N⫹ membranes (Amersham). The CUM1
and CUM26 3⬘ gene-specific cDNA fragments were labeled by random oligonucleotide priming (Feinberg and Vogelstein, 1984). Blots
were hybridized as described by Angenent et al. (1992).
In situ hybridization experiments were performed according to
Cañas et al. (1994). In vitro transcribed RNA probes were labeled
with digoxigenin according to the Boehringer Mannheim labeling kit.
Fragments spanning nucleotides 395 to 862 and 365 to 807 of the
cDNA sequences of CUM1 and CUM26, respectively, were used as
probes.
Analysis of the gp Mutant CUM26 Gene
Figure 7. Summary of Organ Identities in the Four Floral Whorls of
Male and Female Flowers of Wild-Type and Class A, B, and C Cucumber Mutants.
Indeterminacy is indicated by “flower.” WT, wild type.
From the gp mutant, very young floral buds were collected and used
for poly(A)⫹ RNA isolation. One microgram was reverse transcribed
from an oligo(dT) primer by using the Superscript preamplification
system for first strand synthesis (Gibco BRL). The first strand cDNA
was used as a template for polymerase chain reaction (PCR) amplification by using the CUM26-specific primers 5⬘-GGATCGATGGATCCAATGGGAAGAGGGAAAATAG-3⬘ and 5⬘-CCATCGATCCATGGCTCCCAATAGCATAAACATAAAG-3⬘. Subsequently, three independent PCR products were sequenced.
492
The Plant Cell
ACKNOWLEDGMENTS
We thank Jeroen van Arkel for technical assistance and Nilla Pelucchi
for helping with the in situ hybridization experiments. This project
was supported by Bruinsma Seeds BV (currently part of Seminis
Vegetable Seeds) and by the Dutch Department of Commerce.
Received September 12, 2000; accepted December 18, 2000.
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