Sex Determination in the Monoecious Species Cucumber Is Confined to Specific Floral Whorls

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1 The Plant Cell, Vol. 13, , March 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 1 Current address: Dipartimento di Genetica e Biologia dei Microrganismi, Università di Milano, via Celoria 26, Milan, Italy. 2 Current address: Seminis Vegetable Seeds, Inc., State Highway 16, Woodland, CA Current address: Dipartimento di Biologia, Università di Milano, Via Celoria 26, Milan, Italy. 4 To whom correspondence should be addressed. fax 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 influence 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.

2 482 The Plant Cell 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 PIS- TILLATA (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

3 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.

4 484 The Plant Cell C genes are still expressed in primordia that are arrested in development. Phenotype and Molecular Analyses of a Class B Homeotic Mutant 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). 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 morphology 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

5 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 according 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

6 486 The Plant Cell Figure 4. Flower Morphology of the gp Mutant.

7 Sex Determination in Cucumber 487 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 flowers, 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.

8 488 The Plant Cell Figure 5. Flower Morphology of Transgenic Cucumber Plants in Which CUM1 Was Ectopically Expressed or Cosuppressed.

9 Sex Determination in Cucumber 489 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. 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 T 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 T 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 T 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 T 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.

10 490 The Plant Cell 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. 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. Sex Determination 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-

11 Sex Determination in Cucumber 491 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. 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 -GGATCGATGGAT- CCAATGGGAAGAGGGAAAATAG-3 and 5 -CCATCGATCCATGGC- TCCCAATAGCATAAACATAAAG-3. Subsequently, three independent PCR products were sequenced.

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14 Sex Determination in the Monoecious Species Cucumber Is Confined to Specific Floral Whorls Martin M. Kater, John Franken, Kim J. Carney, Lucia Colombo and Gerco C. Angenent Plant Cell 2001;13; DOI /tpc This information is current as of May 1, 2018 References Permissions etocs CiteTrack Alerts Subscription Information This article cites 35 articles, 18 of which can be accessed free at: /content/13/3/481.full.html#ref-list-1 Sign up for etocs at: Sign up for CiteTrack Alerts at: Subscription Information for The Plant Cell and Plant Physiology is available at: American Society of Plant Biologists ADVANCING THE SCIENCE OF PLANT BIOLOGY