Sexual Dimorphism in White Campion: Deletion on the Y Chromosome Results in a Floral Asexual Phenotype

Corresponding author: Ioan Negrutiu, ENS de Lyon, RDP-UMR 9938 CNRS/INRA/ENS, Allée d’Italie 46, Lyon, France., ioan.negrutiu{at}ens-lyon.fr (E-mail)

Communicating editor: W. F. SHERIDAN

	ABSTRACT

TOP ABSTRACT MATERIALS AND METHODS RESULTS DISCUSSION LITERATURE CITED

White campion is a dioecious plant with heteromorphic X and Y sex chromosomes. In male plants, a filamentous structure replaces the pistil, while in female plants the stamens degenerate early in flower development. Asexual (asx) mutants, cumulating the two developmental defects that characterize the sexual dimorphism in this species, were produced by gamma ray irradiation of pollen and screening in the M₁ generation. The mutants harbor a novel type of mutation affecting an early function in sporogenous/parietal cell differentiation within the anther. The function is called stamen-promoting function (SPF). The mutants are shown to result from interstitial deletions on the Y chromosome. We present evidence that such deletions tentatively cover the central domain on the (p)-arm of the Y chromosome (Y2 region). By comparing stamen development in wild-type female and asx mutant flowers we show that they share the same block in anther development, which results in the production of vestigial anthers. The data suggest that the SPF, a key function(s) controlling the sporogenous/parietal specialization in premeiotic anthers, is genuinely missing in females (XX constitution). We argue that this is the earliest function in the male program that is Y-linked and is likely responsible for "male dimorphism" (sexual dimorphism in the third floral whorl) in white campion. More generally, the reported results improve our knowledge of the structural and functional organization of the Y chromosome and favor the view that sex determination in this species results primarily from a trigger signal on the Y chromosome (Y1 region) that suppresses female development. The default state is therefore the ancestral hermaphroditic state.

SPECIES with unisexual flowers usually have bipotential floral buds in which the development of reproductive organs of one or the other sex is arrested or undergoes degenerative processes at stages that vary among both mono- and dioecious plants (DELLAPORTA and CALDERON-URREA 1993 ).

In the dioecious white campion (Silene latifolia = Melandrium album), sex determination is controlled by an active Y chromosome, i.e., an XY system. The sexual dimorphism pattern consists of early arrest of androecial development in pistillate flowers (female plants with 2n = 2x = 22A + XX) and pistil replacement by a filamentous structure in staminate flowers (male plants 2n = 2x = 22A + XY; YE et al. 1991 ; GRANT et al. 1994 ). Thus, unisexuality is achieved through at least two sets of functions affecting early male or female development in flowers of opposite sex that are located on the Y sex chromosome, a gynoecium suppression function (GSF) and a stamen-promoting function (SPF). Genetic studies have suggested that the Y chromosome can be divided into four domains (WESTERGAARD 1958 ) harboring distinct features: (Y1) female suppression, containing the GSF; (Y2) male promoting function(s), containing the SPF; (Y3) male fertility; and (Y4) pseudoautosomal region. Despite renewed interest in white campion research (VYSKOT et al. 1993 ; HARDENACK et al. 1994 ; VEUSKENS et al. 1995 ; DONNISON et al. 1996 ; JANOUSEK et al. 1996 ; SCUTT et al. 1997A ; DI STILIO et al. 1998 ), the relative positions of these domains along the Y chromosome remain unclear. Several reasons need to be mentioned. Mutants corresponding to such Y regions have been reported for Y1 (hermaphroditic flowers) and Y3 (male sterile flowers) only (WESTERGAARD 1958 ; VAN NIGTEVECHT 1966 ; DONNISON et al. 1996 ). No mutants at the SPF locus have been reported so far, which makes the position of the Y2 region purely speculative. In addition, proper banding of the Y chromosome has not been achieved and Y chromosome-specific sequences are not available at present.

Mutants affected in the SPF are needed to demonstrate the physical reality of the locus on the Y chromosome. The specific deletion of the SPF locus should result in a mutant still lacking carpels while having the stamens arrested at the same developmental stage as the stamens of the wild-type female flower (FARBOS et al. 1997 ). We note such mutants as asexual mutants (Figure 1).

View larger version (11K): In this window In a new window Download PPT slide   Figure 1. Alterations of key functions on the Y chromosome in white campion can be generated by pollen irradiation (also see LARDON et al. 1999 , this issue). Suppression or loss of the SPF and preservation of the GSF result in asexual mutant flowers lacking both male and female reproductive organs.

In this article we present data on such a novel type of mutation in Silene, which results from defined deletions on the Y chromosome and exhibits the asexual phenotype. The mutants are named asexua (asx). The main characteristics of the identified asx mutants are (1) they were isolated in the M₁ generation in a dioecious system and in a male diploid genetic background; (2) the mutations correlate with defined deletions on the Y chromosome. A comparative cytogenetic analysis of the GSF (LARDON et al. 1999 , this issue) and SPF chromosomal regions allowed us to tentatively position the SPF within the proximal half of the Y(p)-arm; (3) the mutants are exclusively affected in male organ differentiation and function and are thus developmental- arrest mutants; and (4) the anther arrest occurs at the same stage of early sporogenous/parietal cell differentiation in both female and asx flowers. The data are discussed as evidence that the mutation is functionally equivalent to one of the two key functions ("sex deciding genes") in the sexual determination process, namely, the male-promoting SPF function.

	MATERIALS AND METHODS

TOP ABSTRACT MATERIALS AND METHODS RESULTS DISCUSSION LITERATURE CITED

Plant material and control crosses:
The experiments were performed with pollen from two male genetic backgrounds: M5045 (karyological formula 2n = 26,XY; VEUSKENS et al. 1992 ) and MD1 (2n = 24,XY). The karyotype of M5045 was produced and the extra chromosome pair identified and characterized as an acrocentric (data not shown). The meiotic analysis indicated that the extra chromosomes behaved as bivalents showing no pairing with other chromosomes of the complement (LARDON et al. 1999 , this issue).

Control crosses between standard females (M5006, MR1, 2n = 24,XX) or female MH83 (2n = 26,XX) and M5045 or MD1 males produced abundant seed setting, the progeny being male or female as evaluated on at least a 50-progeny analysis per cross. No phenotypic differences exist between plant series with 2n = 24, 25, or 26 chromosomes.

Mutation induction by means of ⁶⁰Co irradiation of pollen:
Pollen was irradiated at 5–50 Krad (50–500 Gy) and used directly for pollination. Germinating seeds were obtained within the 5–20 Krad range. Altered phenotypes were screened by visual and microscopic observations of floral morphology at specific stages of bud development.

Mutant screening in M₁ generation:
The screening was performed in the M₁ generation to favor the identification of mutations specifically affecting the Y chromosome. Upon irradiation, recessive mutations or deletions would only be visible if Y-linked, while mutations on X or autosomes need to be genetically dominant to be expressed in the M₁. The two asexual mutants, asx1 and asx2, were identified among a first series of 400 M₁ plants analyzed (also see LARDON et al. 1999 , this issue). An additional asexual mutant (asx3) obtained in the MD1 (2n = 24,XY) background from 550 M₁ plants screened has been analyzed morphologically and cytologically and exhibits similar properties as asx1 and asx2 (not shown).

Floral morphology in the wild-type condition:
A detailed description of wild-type flower morphology in white campion is given in FARBOS et al. 1997 . Briefly, in wild-type male flowers, the central conical structure gives rise to a "finger-like projection" without evidence of pistil development. The stamens show normal anther and filament formation. This article focuses on the stages at which an archesporial initial in each anther lobe produces both (1) precursors of sporogenous cells and, subsequently, gametophytic tissues toward the center of the organ and (2) precursors of parietal cells as sporophytic nurse tissues toward the epidermis (FAHN 1990 ).

In wild-type female flowers, five carpels grow in the center of the bud and are united to produce the pistil, surrounded by 10 stamen initials. The stamens at the base of the growing gynoecium are stunted (vestigial anthers), being partially immersed in the nectariferous tissue.

Scanning electron microscopy (SEM) and histological analysis:
The protocol for SEM was as described in VEUSKENS et al. 1992 . For histological observations, flower buds (0.5 to 8 mm long) were fixed in a mixture of 50% (v/v) ethanol, 3.7% (v/v) formaldehyde, and 5% (v/v) acetic acid at room temperature under vacuum for 1 hr, then without vacuum for 24 hr. Flower buds were embedded in paraffin and sections were stained in 0.05% toluidine blue.

Cytological analysis:
Metaphase plates were prepared as described by MOURAS et al. 1989 from root tips of axenic root cultures produced by transformation with Agrobacterium rhizogenes (CIUPERCESCU et al. 1990 ). Transformed lines, indispensable for detailed cytogenetic analysis in asexual mutants that are meiotically defective and unable to produce seeds, were obtained for asx1 and asx2 mutants, but not for asx3. Karyological evaluations are based on the white campion standard karyotype as described by CIUPERCESCU et al. 1990 , the Y chromosome arms being designated (p)- and (q)-arms, respectively.

In situ hybridization:
Three repetitive sequence clones were used as probes in these experiments: 4G12 (2.5 kb), 5E4 (1.2 kb), and X43.1(0.7 kb; BUZEK et al. 1997 ). Probes 4G12 and 5E4 were labeled with Dig-dUTP by nick translation and hybridization was carried out according to the manufacturer's instructions (Biochemica, Boehringer Mannheim, Indianapolis). The hybridization mixture contained 10% dextran sulfate, 50% formamide, 2x SSC, 0.5 mg/ml sonicated salmon sperm DNA and labeled probe to a final concentration of 1 ng/µl. The probe was denatured at 80° for 5 min, loaded on metaphase preparations (50 µl), and again denatured together with the chromosomes at 80° for 1 min. Posthybridization washes were in 2x SSC at 37° (twice), 1x SSC at 65° for 5 min, and 2x SSC (twice) at room temperature. Hybrid molecules were detected with digoxygenin antibodies conjugated to alkaline phosphatase and visualized by color development with BCIP (5-bromo-4-chloro-3-indolyl phosphate) and NBT (nitro blue tetrazolium).

The subtelomeric X43.1 DNA fragment was labeled with biotin-14-dATP by nick translation and the hybridization protocol was according to BUZEK et al. 1997 . The biotin-labeled DNA duplexes were detected with FITC-avidin D complex and the signal was amplified by biotinylated goat antiavidin and FITC-avidin D complex (Vector, Burlingame, CA). The slides were mounted in Vectashield (Vector) containing 1.5 µg/ml propidium iodide. The fluorescent signal was examined with a fluorescent Nikon microscope. The chromosome spreads were photographed and the pictures were processed by Adobe Photoshop 3.0 Power PC program.

Terminology:
The two asexual mutants cannot be analyzed genetically. The term "mutant" is therefore employed to describe a stable altered floral phenotype associated with a chromosomal deletion. The mutants have been vegetatively propagated for more than 5 years with no alteration in phenotype. They are considered asexual mutants because they cumulate the two key developmental blocks that separately characterize the wild-type male and female flowers. Mutations affecting stamen development at later stages should be named sterile mutants. The hermaphroditic mutants (LARDON et al. 1999 , this issue) have bisexual flowers and are called bsx, provided that meiotic stages are attained in both sexes.

	RESULTS

TOP ABSTRACT MATERIALS AND METHODS RESULTS DISCUSSION LITERATURE CITED

Three asexual mutants were identified following pollen irradiation and phenotypic screening in the M₁ generation (see MATERIALS AND METHODS). asx1 and asx2 are derived from the M5045 background, while asx3 is derived from the MD₁ background. asx1 and asx2 are presented in detail here.

The mutant phenotype:
Plant habit, branching, and inflorescence pattern are characteristic of wild-type male plants. Perianth organs are normal, while the development of the pistil and the anthers is arrested very early in floral development [at stages 5 and 6, respectively, according to the nomenclature of FARBOS et al. 1997 and GRANT et al. 1994 ]. Thus the mutants cumulate the two developmental blocks that separately characterize the wild-type male and female flowers. There is no floral organ transformation or conversion. SEM (Figure 2) and histological observations of the two mutants (Figure 3) showed the following:

1. As in the wild-type male flower, pistil formation does not occur; instead a "finger-like projection" develops in the center of the flower. This structure bears no apparent structural or functional similarities with the pistil of wild-type female flowers (Figure 2A vs. D, E, G, I, J, K); the finger-like projection increases steadily in size and remains viable until anthesis.

   
   

   View larger version (140K): In this window In a new window Download PPT slide   Figure 2. Defined stages of reproductive organ development in flower buds of wild-type female and asexual mutants asx2 and asx1. The staging of flower bud development is according to FARBOS et al. 1997 . (A–C) Wild-type female flower. (A) Flower bud at female stage 7.2 showing three stamens undergoing the first step(s) of differentiation: anthers at bilobal stage with short filaments. (B) Stage 8 with enlarged ovary and stamens reaching terminal development (also see Figure 3B). (C) Detail of vestigial anthers of the flower at stage 12 (in bloom). (D–H) Flower development in the mutant asx2. (D) Flower bud at corresponding male stage 7 with two subwhorls of stamens. The outer whorl stamens have bilobal anthers. (E) Stage 9 exhibiting petals, the central "finger-like projection," and stamens at terminal differentiation: bilobal anthers in both inner and outer stamen circles. (F) Detail of the abaxial side of the stamen rudiments showing cell collapse at stage 11. (G) Top view of a flower bud at the same stage: the "finger-like projection" was sectioned. It contains parenchymatous cells only. (H) Flower at stage 12 showing vestigial stamens (arrowheads). (I–M) Flower development in the mutant asx1. (I) Flower bud at male stage 9 with the inner and outer circles of stamens at terminal differentiation (compare with E from above). (J) Bud with detached petals to show the beginning of stamen degeneration (arrow) at early stage 10. (K) Top view of a flower bud at stage 12 (in bloom). Note the atrophy of the stamens. (L) Section through a stamen at the same stage as in J, showing only parenchymatous cells. (M) Detail of the abaxial side of degenerated stamens as shown in K. P, petal; St, stamen, primordium, or vestigial; Ov, ovary. Bar, 100 µm.

   
   

   View larger version (59K): In this window In a new window Download PPT slide   Figure 3. Comparative pattern of floral organogenesis in female (wild-type), asx mutants, and male (wild-type) flower buds with emphasis on the identification of the critical anther developmental arrest point at the stage of initiation of parietal-sporogenous differentiation. The corresponding staging of flower bud development is described in detail in FARBOS et al. 1997 . (A) Temporal sequence of events according to flower bud size and main steps in reproductive organ differentiation as observed in histological preparations (cf. B–I). Female flowers produce a mature and functional pistil, but arrest stamen development after sporogenous cell initials are produced within the anther. Male flowers develop functional anthers and stop pistil formation at primordium stage. The asx mutants cumulate the two developmental blocks. Filled bars indicate the size of the flower buds and the precise histological status at which the arrest in stamen and/or carpel development has occurred. Dotted lines indicate arrested programs. S, sepals; P, petals; St, stamens; C, carpels. (B–I) Histological evaluation of anther arrest in wild-type female (B and C) vs. asx2 and asx1 mutants (D–G) and wild-type male flower (H and I) as control. The upper line (B, D, F, and H) shows whole bud sections (bilobal anthers are arrowed) at female stage 8 and male stage 7, respectively. Note that the sporogenous stage corresponds to larger flower buds in female flowers (cf. A) that produce a normally developing pistil with ovules (arrowhead). The bottom (C, E, G, and I) gives detail from one anther lobe to show the differentiation of sporogenous cells. Sporogenous cell masses are initiated as areas of larger cells with obvious nuclei. Parietal cells (Pa) are visible as one or two layers in male flowers, while absent in female and asx flowers. Bars: B, D, F, H, 100 µm; C, E, G, I, 10 µm.

2. As in the wild-type female flower, the stamens evolve to the point where the anthers reach the bilobal stage, with short, non-elongated filaments (Figure 2A and Figure B vs. D, E, G, I, J and Figure 3B, Figure D, Figure F). The anthers reach a maximum diameter of 150 µm in both wild-type female and asx mutants. Note that in SEM pictures the lobes of the anthers are more prominent in the asx mutants than in female flowers. The stamens in the female flowers are embedded in nectariferous tissues, which are not observed in the two mutants (Figure 2C vs. F, G, K, M). This could explain the apparently more compact aspect of the stamen filament initials in female flowers. To assess more precisely the developmental anatomy of anther arrest, we conducted histological observations.

3. As in the wild-type female flower and contrary to wild-type male flowers, the anthers in asx mutants initiate the formation of sporogenous cells but lack any parietal cell layer (Figure 3C, Figure E, Figure G, Figure I). Sporogenous differentiation is transient, being abruptly arrested at the bilobal stage, followed by differentiation of the sporogenous cells into parenchymatous cells (Figure 2L). Subsequently, the anthers degenerate slowly (Figure 2C, Figure G, Figure H, Figure K, Figure M) and become vestigial.

This evolution is summarized in Figure 3A, which compares female, asx, and male flower development according to bud size (FARBOS et al. 1997 ) to show how the asx mutants cumulate the two developmental blocks. It is important to note that anther arrest in female and asx flowers affects the same cell types (Figure 3C, Figure E, Figure G), but the developmental block occurs in anthers at two different flower bud sizes (i.e., stages) in female vs. asx flowers (Figure 3A, Figure B, Figure D, Figure F, Figure H). Thus, until the point of anther developmental arrest, the asx anthers follow the same pattern of development as those of wild-type male flowers, while the same anther developmental process in female flowers takes a longer time to reach the same histological stage. Such cell fate determination processes, in which the temporal control of gene pathways acts independently of the spatial patterning signals, are known, for example, in Caenorhabditis elegans (EULING and AMBROS 1996 ). Finally, the carpel developmental arrest in asx mutants is identical to that occurring in the wild-type male flower (Figure 3D, Figure F, Figure H).

Cytogenetical analysis of the asexual mutants:
The two asx mutants described here derive from the M5045 male background (see MATERIALS AND METHODS) and therefore have 2n = 25,XY (Table 1). That the additional chromosome has no influence on the phenotypic sex was established in previous experiments with haploid and spontaneously doubled haploids (VEUSKENS et al. 1992 ) in this genotype, in parallel experiments with certain bsx mutants (LARDON et al. 1999 , this issue), and with additional data detailed in MATERIALS AND METHODS.

The asexual mutants exhibit defined deletions on the Y chromosome: Metaphase plates in the control male line (Figure 4A) and asexual mutants asx2 (Figure 4B) and asx1 (Figure 4C) were analyzed for arm ratio, centromeric index, and relative length of all chromosomes. As no changes were registered in the autosomes or the X chromosome, Table 1 presents data for the Y chromosome alone, in which significant changes in chromosomal length occurred. In asx2 the deletion affected ~12% of the chromosome length, while in asx1 the deletion covered ~20% of the chromosome length. Because both mutants had the same phenotype, the genetic information triggering anther differentiation in male flowers should be located within the 12% deleted area from the Y chromosome in asx2. Because the Y chromosome is metacentric and the asx mutants are arrested much earlier than the meiotic stage, we were unable to specifically assign, by arm ratio measurements alone, the deletion to the (p)- or the (q)-arm. For this purpose, the use of molecular marker mapping to the Y chromosome was attempted.

View larger version (33K): In this window In a new window Download PPT slide   Figure 4. Metaphase plates from wild-type male (A), asexual mutants asx2 (B), and asx1 (C). The sex chromosomes are marked as the X chromosome (arrowhead) and the Y chromosome (arrow). The Y chromosomes in the mutants are deleted (cf. Table 1).

Choice of physical markers for Y deletion mapping: Differential screening performed on genomic libraries constructed from male plant DNA (essentially HindIII and BamHI fragments) resulted in the identification of ~30 clones that, when tested by Southern blot analysis on 25 different male genotypes, were characterized as polymorphic rather than specific. Five such clones, 2G1, 4G12, 5E4, 6D8, and 4E7 recognized sequences present on autosomes and on the Y chromosome when analyzed by in situ hybridization (I. FARBOS, A. MOURAS and J. VEUSKENS, unpublished results). Sequences with similar properties have been reported in humans (CHARLESWORTH 1991 ; FOOTE et al. 1992 ). These five clones were defined as Y-located, none of them being Y-specific. Clones 4G12 and 5E4 were chosen for further characterization of the asx mutants (Figure 5A and Figure B). In addition, the subtelomeric probe X43.1 was tested to identify potentially terminal deletions, because it was shown that this repetitive sequence recognizes the pseudoautosomal arm of the Y chromosome. Our tests revealed that X43.1 is also polymorphic, as at least two types of wild-type Y chromosomes exist with respect to X43.1: one class corresponding to the described arm asymmetry of X43.1 (BUZEK et al. 1997 ), the second having X43.1 sequences at both ends of the Y (Figure 5C).

View larger version (53K): In this window In a new window Download PPT slide   Figure 5. Y-deletion mapping based on Y chromosome hybridization pattern in wild-type male, asx mutants (4G12, 5E4, and X43.1 repetitive sequence probes), and one bsx mutant (X43.1 probe). Twenty to 60 plates were analyzed per genotype. Signal frequency was evaluated at 35 to 85% in the three lines for 4G12, at 70% for 5E4 in the wild-type male line, and at 90% for X43.1. (A) Detail of the 4G12 signal on individual Y chromosomes from wild-type male, asx2 and asx1 mutants (in this order from left to right) and (B) for 5E4 clone. (C–F) Detail of the X43.1 signal on X and Y chromosomes from wild type (C), bsx3 (D), asx1 (E), and asx2 (F). C, E, and F correspond to mitotic chromosomes; D corresponds to a meiotic preparation.

The deletions in mutants asx1 and asx2 affect the same arm of the Y chromosome: Y mapping with the repetitive DNA sequences was performed to estimate the orientation of the Y deletions and to determine the chromosomal location of the deletions. In particular, probe X43.1 was tested in wild-type males, two asx mutants, and one bsx mutant (bsx3; LARDON et al. 1999 , this issue). Because these lines share a Y chromosome derived from the same paternal genotype, M5045, the relative positions of the deletions in asx and bsx mutants could be directly compared.

In situ hybridization experiments were performed on metaphase chromosomes of wild type, asx2, and asx1. Using 4G12 insert as probe, homologous sequences were identified on the Y chromosome of the wild type and both asx mutants, namely, on the nondeleted arm (Figure 5A). In contrast, the 5E4 insert used as probe (Figure 5B) produced signals on the wild-type Y chromosome only, no signal being detected on the deleted Y chromosome of the asexual mutants. Comparison of results using the two probes indicated that in both mutants the deletions affected the central region of the same Y arm.

The results of the in situ hybridization with the subtelomeric probe X43.1 in wild type, asx mutants (mitotic preparations), and mutant bsx3 (meiotic preparation; LARDON et al. 1999 , this issue) are presented in Figure 5C–F and show that the X43.1 signal is present at both ends of the Y chromosome in wild type and asx mutants, but the signal is missing in bsx3 at the level of the differential (p)-arm. This indicates that the deletion in both asx mutants is interstitial, confirming the results obtained with the 5E4 probe. Assuming that GSF and SPF loci are located on the same arm of the Y, and because the two classes of mutants, bsx and asx, exhibit mutually exclusive phenotypes, two models can be proposed concerning the linkage configuration between the two chromosomal regions (Figure 6). Both models take into account the fact that the largest Y deletion having generated a bsx phenotype covers 51% of the Y(p)-arm (mutant bsx1; LARDON et al. 1999 , this issue). One model predicts relatively tight linkage (Figure 6A; DONNISON et al. 1996 ), the GSF chromosomal region mapping just distal to the breakpoint pY51 in bsx1 and being closely located to a function such as SPF. If this model is correct, the two largest deletions we obtained, bsx1 (51%) and asx1 (40%), could almost entirely cover the (p)-arm. The other model (Figure 6B) assigns SPF to a location proximal to the "pY51" position and GSF toward the tip of the (p)-arm. In the accompanying article (LARDON et al. 1999 ), we present preliminary evidence in favor of the (p)-arm terminal location of GSF. This would imply that linkage of GSF with SPF on the (p)-arm is loose. Overlapping deletions should generate, depending on the exact nature of the male function involved (SPF and possibly other male genes), phenotypic females or male sterile hermaphroditic mutants, respectively. The former ones cannot be detected by our visual screening protocol.

View larger version (10K): In this window In a new window Download PPT slide   Figure 6. Diagram of the Y chromosome (p)-arm containing the two key functions that control sexual dimorphism in white campion: the GSF and the SPF. Two working hypotheses are proposed for the location of GSF and SPF loci with respect to the largest deletions affecting GSF (51%) and SPF (40%). p51 represents a breakpoint that delimits the GSF and SPF domains. (A) The "tight" linkage model and (B) the "loose" linkage model. Within the respective domains, GSF and SPF loci can occupy other positions than those indicated. The heterochromatic regions are based on G-banding data.

	DISCUSSION

TOP ABSTRACT MATERIALS AND METHODS RESULTS DISCUSSION LITERATURE CITED

In this article we characterize two independent mutants with interstitial deletions on the Y chromosome that exhibit an identical asexual phenotype resulting from the disruption of early stamen differentiation. Because both asx mutants have deleted Y chromosomes and whorl 4 is unaffected, being genuinely blocked in male flowers, it is considered that the mutations have occurred in a male genetic background and affect exclusively anther development (also see below). Both asx mutants are phenotypically very stable, with no variation in their flower formula (S₅P₅a₁₀C₀) over several years of vegetative propagation.

The asx mutants are developmental arrest mutants of early anther differentiation because a gene(s) controlling a defined developmental stage of anther formation is disrupted in the two mutants. This results in specific arrest of stamen differentiation at the stage of sporogenous/parietal cell-type initiation, equivalent to that observed in female flowers (XX configuration). Because anther arrest occurs at the same stage of anther formation and affects the same tissue type in both female and asexual flowers, three conclusions can be drawn: (1) the observed events are most likely part of the same developmental pathway; (2) the initiation, but not the maintenance, of the sporogenous cell differentiation can occur in the absence of parietal tissues, and (3) the sporogenous/parietal cell differentiation appears to be the earliest male function encoded by the Y chromosome, and we postulate that it is this function that has been affected in both asx mutants showing deletions on the Y chromosome. It follows that this function defines the sexual dimorphism in the Silene male program and therefore should correspond to the SPF locus. However, only by cloning SPF can one exclude the possibility that additional genes somewhere else in the genome are involved in the asx phenotype.

In summary, asx1 and asx2 represent a novel class of mutants in plants that affect the first stages of sporogenous/parietal tissue specialization in the anther. The common ancestry of the sporophytic (parietal) and gametophytic (sporogenous) cell types implies a tight control on positional information and cellular recognition. This stage is therefore critical in anther development as it marks a transition from prepattern to pattern formation. No genes involved in sporogenous/parietal cell differentiation have yet been reported in plants. Interestingly, the smut fungus Microbotryum violaceum (RUDDAT et al. 1991 ; SCUTT et al. 1997B ) can complement such a developmental defect as female-infected plants (XX configuration) develop morphologically normal stamens. Whether the fungus is able to complement the SPF mutation remains to be answered in future experiments. We have made use of asx mutants to study early differentiation events in reproductive organ formation by cloning stamen-specific, premeiotically expressed genes in this species (BARBACAR et al. 1997 ) and have identified, for example, a parietal layer-specific marker gene (HINNISDAELS et al. 1997 ; ROBERTSON et al. 1997 ). Such genes represent the missing link within the frame of regulatory mechanisms that control tissue/cellular specialization downstream of the flower pattern genes.

Asexual mutants of the type described here have not been reported until now in plants, but WESTERGAARD 1958 postulated the existence of a Y-borne male "promoting" function(s) that was assigned by default to a broad pericentromeric Y region (Figure 7A). The asx mutants support the existence of such a domain and provide the first experimental evidence that a putative SPF gene(s) is located in a defined region of the Y chromosome (Figure 7B) that has been narrowed down to 24% of the corresponding arm length.

View larger version (22K): In this window In a new window Download PPT slide   Figure 7. Functional domains on the Y chromosome according to WESTERGAARD 1958 (A) and this work (B). The analysis of the two asexual mutants asx1 and asx2 indicates that the Y2 region is restricted to the central domain of the (p)-arm of the Y chromosome. The present work locates region Y1 according to results of LARDON et al. 1999 (this issue) and leaves open the assignment of region Y3.

The comparative analysis of Y chromosome breakpoint location in asx and bsx mutants is very useful because the two classes of mutants exhibit mutually exclusive phenotypes. Despite a lack of Y-specific markers, we have tentatively located the SPF locus within the central area of the (p)-arm. However, we cannot exclude the possibility that the interstitial deletions have occurred on the (q)-arm. Similarly, and despite being in agreement with the results of WESTERGAARD 1958 , our results on the terminal location of the GSF chromosomal region on the (p)-arm need additional assessment. Note, however, that our proposed GSF location is supported by indirect evidence, such as higher rates of bsx mutations as compared with asx mutations obtained at low -ray dosages (LARDON et al. 1999 , this issue), together with higher incidences of single vs. double break deletions reported to occur at low irradiation dosages (LEE and KAMRA 1981 ; LEFRANCOIS et al. 1989 ) or the efficient healing of terminal deficiencies as reported in maize, wheat, and Drosophila (MCCLINTOCK 1942 ; BIESSMANN 1990 ; TSUJIMOTO 1993 ). Attempts to map the (p)-arm have also been made by DONNISON et al. 1996 using Y-deletion mutants with bsx or male sterile phenotypes and three Y-linked DNA markers. As in our case, the proper assignment of the GSF and SPF-like chromosomal regions requires further tests. As a matter of fact, the reported deletions (DONNISON et al. 1996 ) were not characterized cytogenetically, the phenotypes of male sterile mutants were not analyzed histologically, and three bsx-type mutants apparently carrying the largest deletions and lacking those markers were male fertile. Further work with bsx and asx mutants should clarify these points and allow us to solve the linkage configuration between the GSF and SPF chromosomal regions (Figure 6 and Figure 7), an important issue in the understanding of both functional and evolutionary aspects of the XY system in this species.

The Y-deleted asx mutants described here show that altering SPF, the earliest putative stamen differentiation factor(s) located on the Y, can result in a mutated phenotype in an XY configuration. This implies that the SPF has no functional counterpart elsewhere in the genome. The slow regression within the anther of sporogenous cell initials into parenchymatous cells, apparently without cell lysis, tends to support this view. The fact that GSF and SPF chromosomal regions behave as "linked" dominant traits constitutes the basis of the chromosomal mechanism of sex determination in white campion. The classical Y/X formula f^Sm^F/f^fm^s (CHARLESWORTH 1996 ), corresponding to current models on evolution of separate sexes and generation of "primitive" sex chromosomes, finally becomes f^Sm^F/- - (also see LARDON et al. 1999 , this issue).

In conclusion, stamen arrest in asx and female flowers is due to the absence of one or more developmental cues in the anther differentiation pathway resulting from the SPF. We propose that the SPF is encoded within the proximal half of the (p)-arm central domain of the Y chromosome. SPF is absent from the female XX genomic configuration (lack of expression or physical loss) and is deleted in the asx mutants. The results indicate that male "determination" does not result from a trigger signal on the Y chromosome, in the sense that stamen identity and initiation are taking place in XX plants, the role of SPF being to stimulate the completion of stamen development. Instead, sexual dimorphism in Silene is a process that involves both a subversion (a "diversion") of the program of female development in male flowers and the functional or physical interruption of the male differentiation pathway in female flowers. The default state in Silene is therefore the ancestral hermaphroditic state. Our results contribute to specification of how white campion has undergone—among a variety of evolutionary options (CHARLESWORTH 1991 )—an early and dramatic set of developmental changes affecting reproductive organ differentiation and leading to unisexuality.

	FOOTNOTES

¹ These authors contributed equally to this work.

	ACKNOWLEDGMENTS

The authors are indebted to Deborah Charlesworth, Françoise Monéger, Gwyneth Heckel, Mark Cock, and Charlie Scutt for critical reading of the manuscript. The work was partly supported by the Nationaal Fonds voor Wetenschappelijk Onderzoek project G.2148.94 to I.N., by the grant agency of the Czech Republic (521/99/0609) to B.V., and by an Instituut Wetenschappelijk Onderzoek Nijverheid en Landbouw grant to J.V.

Manuscript received May 11, 1998; Accepted for publication December 8, 1998.

	LITERATURE CITED

TOP ABSTRACT MATERIALS AND METHODS RESULTS DISCUSSION LITERATURE CITED

BARBACAR, N., S. HINNISDAELS, I. FARBOS, F. MONÉGER, and A. LARDON et al., 1997  Isolation of early genes expressed in reproductive organs of the dioecious white campion (Silene latifolia) by subtraction cloning using an asexual mutant. Plant J. 12:805-817[Medline].

BIESSMANN, H., 1990  Addition of telomere-associated HeT DNA sequences "Heals" broken chromosome ends in Drosophila.. Cell 61:663-673[Medline].

BUZEK, J., H. KOUTNIKOVA, A. HOUBEN, K. RIHA, and B. JANOUSEK et al., 1997  Isolation and characterization of X chromosome-derived DNA sequences from a dioecious plant Melandrium album.. Chromosome Res. 5:57-65[Medline].

CHARLESWORTH, B., 1991  The evolution of sex chromosomes. Science 251:1030-1032[Abstract/Free Full Text].

CHARLESWORTH, B., 1996  The evolution of chromosomal sex determination and dosage compensation. Curr. Biol. 6:149-162[Medline].

CIUPERCESCU, D. D., J. VEUSKENS, A. MOURAS, D. YE, and M. BRIQUET et al., 1990  Karyotyping Melandrium album, a dioecious plant with heteromorphic sex chromosomes. Genome 33:556-562.

DELLAPORTA, S. L. and A. CALDERON-URREA, 1993  Sex determination in flowering plants. Plant Cell 5:1241-1251[Free Full Text].

DI STILIO, V. S., R. V. KESSELI, and D. L. MULCAHY, 1998  A pseudoautosomal random amplified polymorphic DNA marker for the sex chromosomes of Silene dioica. Genetics 149:2057-2062[Abstract/Free Full Text].

DONNISON, I. S., J. SIROKY, B. VYSKOT, H. SAEDLER, and S. R. GRANT, 1996  Isolation of Y chromosome-specific sequences from Silene latifolia and mapping of male sex-determining genes using representational difference analysis. Genetics 144:1893-1901[Abstract].

EULING, S. and V. AMBROS, 1996  Reversal of cell fate determination in Caenorhabditis elegans vulval development. Development 122:2507-2515[Abstract].

FAHN, A., 1990 The stamen, pp. 430–457 in Plant Anatomy. Pergamon Press, Oxford.

FARBOS, I., M. OLIVEIRA, I. NEGRUTIU, and A. MOURAS, 1997  Sex organ determination and differentiation in the dioecious plant Melandrium album (Silene latifolia): a cytological and histological analysis. Sex. Plant Reprod. 10:155-167.

FOOTE, S., D. VOLLRATH, A. HILTON, and D. C. PAGE, 1992  The human Y chromosome: overlapping DNA clones spanning the euchromatic region. Science 258:60-66[Abstract/Free Full Text].

GRANT, S., B. HUNKIRCHEN, and H. SAEDLER, 1994  Developmental differences between male and female flowers in the dioecious plant Silene latifolia.. Plant J. 6:471-480.

HARDENACK, S., D. YE, H. SAEDLER, and S. GRANT, 1994  Comparison of MADS box gene expression in developing male and female flowers of the dioecious plant white campion. Plant Cell 6:1775-1787[Abstract/Free Full Text].

HINNISDAELS, S., A. LARDON, N. BARBACAR, and I. NEGRUTIU, 1997  A floral third whorl-specific marker gene in the dioecious species white campion is differentially expressed in mutants defective in stamen development. Plant Mol. Biol. 35:1009-1014[Medline].

JANOUSEK, B., J. SIROKY, and B. VYSKOT, 1996  Epigenetic control of sexual phenotype in a dioecious plant, Melandrium album.. Mol. Gen. Genet. 250:483-490[Medline].

LARDON, A., S. GEORGIEV, A. AGHMIR, G. LE MERRER, and I. NEGRUTIU, 1999  Sexual dimorphism in white campion: complex control of carpel number is revealed by Y chromosome deletions. Genetics 151:1173-1185[Abstract/Free Full Text].

LEE, C. L. and O. P. KAMRA, 1981  The pattern of radiation-induced transmissible aberrations in a human cell culture. Hum. Genet. 57:380-384[Medline].

LEFRANÇOIS, D., W. AL ACHKAR, A. AURIAS, J. COUTURIER, and A. M. DUTRILLAUX et al., 1989  Chromosomal aberrations induced by low-dose -irradiation: study of R-banded chromosomes of human lymphocytes. Mutat. Res. 212:167-172[Medline].

MCCLINTOCK, B., 1942  The fusion of broken ends of chromosomes following nuclear fusion. Proc. Natl. Acad. Sci. USA 28:458-463[Free Full Text].

MOURAS, A., I. NEGRUTIU, M. HORTH, and M. JACOBS, 1989  From repetitive DNA sequences to simple-copy gene mapping in plants by in situ hybridization. Plant Physiol. Biochem. 27:161-168.

ROBERTSON, S. R., L. YI, C. P. SCUTT, M. E. WILLIS, and P. M. GILMARTIN, 1997  Spatial expression dynamics of the Men-9 gene family delineates the third floral whorl in male and female flowers of dioecious Silene latifolia. Plant J. 12:155-168[Medline].

RUDDAT, M., J. KOKONTIS, L. BIRCH, E. D. GARBER, and K.-S. CHIANG et al., 1991  Interactions of Microbotryum violaceum (Ustilago violacea) with its host plant Silene alba.. Plant Sci. 80:157-165.

SCUTT, C. P., Y. KAMISUGI, F. SAKAI, and P. M. GILMARTIN, 1997a  Laser isolation of chromosomes demonstrates high sequence similarity between the X and Y sex chromosomes of dioecious Silene latifolia. Genome 40:705-715[Medline].

SCUTT, C. P., L. YI, S. R. ROBERTSON, M. E. WILLIS, and P. M. GILMARTIN, 1997b  Sex determination in dioecious Silene latifolia: Y chromosome and Ustilago violacea-mediated effects during dioecious flower development. Plant Physiol. 122:969-979.

TSUJIMOTO, H., 1993  Molecular cytological evidence for gradual telomere synthesis at the broken chromosome ends in wheat. J. Plant Res. 106:239-244.

VAN NIGTEVECHT, G., 1966  Genetic studies in dioecious Melandrium: I. Sex-linked and sex-influenced inheritance in Melandrium album and Melandrium dioicum. Genetica 37:281-306.

VEUSKENS, J., D. YE, D. CIUPERCESCU, P. INSTALLÉ, and H. A. VERHOEVEN et al., 1992  Sex determination in Melandrium album: androgenic embryogenesis requires the presence of the X-chromosome. Genome 35:8-16.

VEUSKENS, J., D. MARIE, S. C. BROWN, M. JACOBS, and I. NEGRUTIU, 1995  Flow sorting of the Y sex chromosome in the dioecious plant Melandrium album.. Cytometry 21:363-373[Medline].

VYSKOT, B., A. ARAYAS, J. VEUSKENS, I. NEGRUTIU, and A. MOURAS, 1993  DNA methylation of sex chromosomes in a dioecious plant, Melandrium album.. Mol. Gen. Genet. 239:219-224[Medline].

WESTERGAARD, M., 1958  The mechanism of sex determination in dioecious flowering plants. Adv. Genet. 9:217-281[Medline].

YE, D., M. OLIVEIRA, J. VEUSKENS, Y. WU, and P. INSTALLÉ et al., 1991  Sex determination in the dioecious Melandrium: the X/Y chromosome system allows complementary cloning strategy. Plant Sci. 80:93-106.

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