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Annals of Botany 86: 211±221, 2000 doi:10.1006/anbo.2000.1201, available online at http://www.idealibrary.com on B OTA N I CA L B R I E F I N G Boys and Girls Come Out to Play: The Molecular Biology of Dioecious Plants C H A R L E S A I N S WO R T H * Plant Molecular Biology Laboratory, Department of Biology Sciences, Imperial College at Wye, Ashford, Kent TN25 5AH, UK Received: 14 February 2000 Returned for revision: 28 March 2000 Accepted: 25 April 2000 The majority of the world's ¯owering plants are hermaphrodite but many of them encourage cross pollination by means of spatial or temporal separation of eggs and pollen, or by genetically-controlled physiological incompatibility. A minority of species has taken the avoidance of self-pollination to its logical conclusion by evolving two distinct and sexually dierent forms (dioecy). In a very small number of plants, dioecy has been accompanied by the development of sex chromosomes. From the study of the development of male and female ¯owers of dierent species it is clear that there is no common underlying mechanism and that sex determination systems leading to dioecy have originated independently many times in evolution. This Botanical Brie®ng highlights new information from recent molecular # 2000 Annals of Botany Company approaches in the study of dioecy. Key words: Review, sex determination, dioecy, sex chromosome. I N T RO D U C T I O N The great majority of ¯owering plants (around 90 %) produce ¯owers which are `perfect'; these hermaphroditic ¯owers are both staminate (with stamens) and pistillate (with one or more carpels). Although the terms `male' and `female' should be reserved for gametes or the direct producers of gametes ( for example, male and female animals), for convenience we can regard the stamens (collectively the androecium) as the `male' organs of a ¯ower and the carpels (collectively the gynoecium) as the `female' organs. The situation in higher plants contrasts strikingly with that in animals where most species are unisexual with male and female gametes produced by dierent individuals. Ten percent or so of plant species (Yampolsky and Yampolsky, 1922) have evolved ¯oral unisexuality as spatial separation of their ¯owers. This can be manifested as monoecy, where the male and female organs are carried on separate ¯owers on the same plant, or dioecy, where male and female ¯owers are carried on separate male (staminate) or female ( pistillate) individuals (Fig. 1). A number of other sex states or breeding systems are found (Fig. 1), which may be intermediates during the evolution of full unisexuality or may be stable forms. These are gynodioecy, in which populations are composed of female and hermaphroditic plants (e.g. Plantago coronopus; Koelewijn and van Damme, 1996), androdioecy, in which populations are composed of male and hermaphroditic plants (e.g. Datisca glomerata; Liston et al., 1990), trioecy or subdioecy, in which populations are composed of male, female and hermaphroditic plants (e.g. Pachycereus pringlei; Fleming et al., 1994), gynomonoecy, in which plants carry female and hermaphroditic ¯owers (e.g. Poa spp.; * E-mail [email protected] 0305-7364/00/080211+11 $35.00/00 Anton and Connor, 1995), andromonoecy, in which plants carry male and hermaphroditic ¯owers (e.g. Cucumis melo; Rosa, 1928), and trimonoecy, in which plants carry male, female and hermaphroditic ¯owers (e.g. Dimorphotheca pluvialis; Correns, 1906). Whilst gynodioecy, gynomonoecy, and trimonoecy are relatively common, androdioecy, trioecy and trimonoecy are rare. Unisexual plant species and their mixed forms appear to be distributed throughout the ¯owering plant families (around three quarters of families include dioecious species) suggesting independent evolutionary events. However, the incidence of unisexuality is not evenly spread throughout the plant kingdom; dioecy is particularly prevalent in the families Menispermaceae, Myristicaceae, Monimiaceae, Euphorbiaceae, Moraceae, Cucurbitaceae, Anacardiaceae and Urticaceae, and appears to be rather more common among dicot genera than among monocot genera (Renner and Ricklefs, 1995). A number of agronomically important plants are monoecious or dioecious (Table 1). This Botanical Brie®ng will focus primarily on dioecy. E VO L U T I O N O F D I O E C Y Two fundamental questions arise concerning the evolution of unisexuality in plants. These relate to the forces which have driven the evolution of unisexuality and the nature of the evolutionary route. Monoecy and dioecy at ®rst sight appear to be quite dierent animals; dioecy prevents intraindividual self-pollination absolutely, while monoecy merely prevents intra-¯ower self-pollination but not intraindividual self-pollination. In terms of the driving force, it is tempting to consider dioecy as simply the most extreme mechanism which avoids the deleterious eects of inbreeding. In plants, the basic # 2000 Annals of Botany Company 212 AinsworthÐDioecy in Plants F I G . 1. Sex strategies in plants. Those in boxes represent the mixed states. T A B L E 1. Monoecious and dioecious plant species of agronomic importance Monoecious species Castor bean (Ricinus communis) Cucumber (Cucumis sativus) Fig (Ficus carica) Hazelnut (Corylus spp.) Maize (Zea mays) Melon (Cucumis melo) Oil palm (Elaeis guineensis) Walnut (Juglans regia) Dioecious species Asparagus (Asparagus ocinalis) Cloudberry (Rubus chamaemorus) Date palm (Phoenix dactylifera) Hemp (Cannabis sativa) Hop (Humulus lupulus) Kiwifruit (Actinidia deliciosa) Mistletoe (Viscum album) Papaya (Carica papaya) ¯ower pattern has been modi®ed in a number of ways such that sexual reproduction involving outcrossing, and the consequent ¯ow of genetic variation derived from mutation, is favoured (Darwin, 1876). The mechanisms in plants which promote outcrossing include the temporal separation of the maturation of the male and female organs within an otherwise perfect ¯ower (dichogamy; reviewed in Bertin and Newman, 1993), self-incompatibility mechanisms, both sporophytic (reviewed by Nasrullah and Nasrullah, 1993) Pistachio (Pistacia vera) Poplar (Populus spp.) Spinach (Spinacia oleracea) Willow (Salix spp.) Yam (Dioscorea spp.) and gametophytic (reviewed by Newbegin et al., 1993), where there is genetic control over the possible fertilization events, and dioecy, the spatial separation of the sexual organs on separate plants. Were the promotion of outcrossing the sole selective force driving dioecy, one might expect more than 4 % of plant species to be dioecious; in reality, many plant species, particularly annuals with short life cycles (Barrett et al., 1996), are hermaphroditic and sel®ng. Whilst there is AinsworthÐDioecy in Plants clearly a case for dioecy having evolved as an inbreeding avoidance mechanism for some plant species, and particularly so in the case of animal pollinated dioecious species (Freeman et al., 1997), a second mechanism, that of sexual specialization (the dierential selection on male and female aspects of reproduction), is equally, if not more important (Darwin, 1876; Freeman et al., 1997). In hermaphroditic plants, pollen and ovule production may limit each other's production whereas separation of the sexes may enable resources to be allocated more eciently. Resource allocation can dier with respect to the structures of male and female ¯owers, the structure of the in¯orescence and the distribution of male and female ¯owers or in¯orescences within a plant. The structure of the ¯ower is clearly important in relation to pollen distribution and reception and in many hermaphroditic plant species ¯owers have evolved dramatically to suit particular pollination mechanisms. In wind pollinated plants (where there is a strong correlation with dioecy; Renner and Ricklefs, 1995), sexual specialization of male and female ¯owers is common. Staminate ¯owers are often borne on long, pendulous and ¯exible in¯orescences which aid pollen dispersal, while female ¯owers and in¯orescences are generally more rigid and have feathery stigmas (Faegri and Pijl, 1971). The distribution of ¯owers is also important and male ¯owers tend to be borne on slender branches with female ¯owers on large, more rigid branches (Faegri and Pijl, 1971). Similarly, the strong correlation of the incidence of dioecy with climbing growth habit may well have arisen because it is advantageous to have fruit-bearing ¯owers on plants with stronger stems and pollen-bearing ¯owers on thin stems (Renner and Ricklefs, 1995). Theoretical results from modelling the evolution of dioecious populations suggest that resource allocation and the avoidance of inbreeding are not alternatives and that they are probably both involved whenever a dioecious species evolves from a hermaphroditic species (Charlesworth and Guttman, 1999). There are three possible routes to dioecy: from a hermaphrodite, via monoecy, and via distyly. The evolution of a dioecious species directly from a hermaphroditic species is considered unlikely since the occurrence and establishment of two independent mutations, one for male sterility and one for female sterility, must occur simultaneously and the mutant genes (or multiple loci) must be tightly linked so that the generation of hermaphrodites does not occur by recombination (Lewis, 1942; Ross, 1978). It is far more likely (see Charlesworth and Guttman, 1999, for review) that dioecy has evolved through gynodioecy as an intermediate (the coexistence of females and hermaphrodites) or through androdioecy (the coexistence of males and hermaphrodites). In both cases, the initial step is the establishment of a single mutant form (Charlesworth and Charlesworth, 1978). Androdioecy is very rare and the relatives of most, if not all, androdioecious species are dioecious (Charlesworth and Guttman, 1999). In androdioecy, the ®rst mutation must cause total or partial female sterility. It is likely that androdioecious populations very quickly evolve into dioecy and that those cases of plant species where there is good evidence for androdioecy (Datisca glomerata, Mercurialis 213 annua) are the result of the breakdown of full dioecy (Charlesworth and Guttman, 1999). Gynodioecy in plants is far more common than androdioecy and must be considered to be a more likely intermediate step to dioecy than is androdioecy. Dioecious species commonly have gynodioecious relatives (Charlesworth and Charlesworth, 1978). A single gene mutation or cytoplasmic male sterility system generating females (causing male sterility) will spread through a population if mutant plants are ®tter than the hermaphrodites (Lewis, 1941; Ross and Shaw, 1971; Lloyd, 1974). In a population suering inbreeding depression, such a mutation will also spread, even if the mutant is no ®tter (Valdeyron et al., 1973; Lloyd, 1975; Charlesworth and Charlesworth, 1978). The evolution of a gynodioecious population to a dioecious one requires a second mutation, causing female sterility in hermaphrodites and generating males. There is good evidence that increased maleness in the hermaphrodites will be advantageous because of the increased availability of ovules ( provided by the females) (Charlesworth and Guttman, 1999). The second evolutionary route to dioecy is through monoecy and has been implicated in the evolution of a number of dioecious species including Mercurialis (Westergaard, 1958; Charlesworth and Charlesworth, 1978). Dioecy is more frequently associated with monoecy than with hermaphroditism (Lewis, 1942). Although monoecy is a developmental modi®cation rather than a genetic separation as is the case for dioecy, a monoecious population could evolve into a dioecious one through a series of mutations which alter the ratio of male to female ¯owers (Charlesworth and Charlesworth, 1979). One of the most interesting examples is that of the wasp pollinated dioecious ®gs (Ficus spp.) of tropical rain forests. The dioecious species are usually found in the understorey whereas monoecious species inhabit the canopy (Harrison, 1996). The dioecious ®gs have clearly evolved from monoecious species but the mechanism itself is not clear. Although inbreeding avoidance is a possibility, recent evidence implicates the pollinating wasp, either involving a reduction of the impact of parasitic wasps on the pollinating wasp (Kerdelhue and Rasplus, 1996) or by maintenance of the pollinating wasp population (Kameyama et al., 1999). Where dioecy has evolved from monoecy, one prediction would be that the species might show some sex lability and that under certain environmental conditions ¯owers of the incorrect sex will be produced (Freeman et al., 1997). A number of dioecious species do show lability of sex (see below). The third possible route to dioecy is via distyly which describes the condition where individuals are polymorphic for style and anther positions as two dierent hermaphroditic ¯oral types. Distyly has probably arisen to promote pollen dispersal (and therefore outcrossing) and is often associated with self incompatibility. Specialization of one type for maleness and the other for femaleness may progress to full dioecy (Lloyd, 1979). Many attempts have been made, using morphological and molecular systematics, to reconstruct the early evolutionary history of the 250 000 or so species of ¯owering 214 AinsworthÐDioecy in Plants plants. Recent sequencing analyses suggest strongly that Amborella trichopoda, an evergreen dioecious shrub endemic to New Caledonia, is the ®rst branch of angiosperm evolution (Parkinson et al., 1999). It is tempting to speculate that the ®rst angiosperms were dioecious and that this state has then evolved into the hermaphrodites which dominate the present ¯ora and which have `reverted' to generate the unisexual species. However, a dioecious ancestral angiosperm seems unlikely and, more probably, a transition from hermaphroditism to dioecy occurred on the branch leading to Amborella. Dioecy is considered by Charlesworth and Guttman (1999) to have evolved more than 100 times to account for the 160 plant families which include dioecious species. S E X C H RO M O S O M E S In a small number of plant species the transition from hermaphroditism has been followed by the evolution of sex chromosomes, which presumably have evolved as a consequence of the need to limit recombination between the dierent sex determining genes. Heteromorphic sex chromosomes in higher plants were ®rst detected in Rumex acetosa and Silene dioica (Melandrium rubrum) by Kihara and Ono (1923) and Blackburn (1923). Although sex chromosomes have been claimed for nearly a hundred plant species, the number of authenticated sex chromosome systems is at least an order of magnitude smaller (Parker, 1990). Plant sex chromosomes are probably of relatively recent origin, in contrast to the situation in animals, and thus aord the opportunity of investigating recent chromosome evolution. Although sex chromosomes in ¯owering plants are rare, plants have evolved systems which are analogous to the main chromosomally-based systems in animals. For example, the Silene species have an active Y system similar to that in mammals where the Y chromosome acts as a maleness enhancer as well as suppressing the gynoecium (see Grant et al., 1994b, for review). The Rumex acetosa group has a Drosophila-type X/autosome dosage system in which the primary sex determination is independent of the presence or absence of the Y chromosomes and is controlled by whether the X:autosome ratio is 1.0 or more (resulting in females) or is 0.5 or less (resulting in males) (see Ainsworth et al., 1999, for review). Dierentiated sex chromosomes have been established clearly in only six families, representing about eight species and two major species groups. In the Cannabidaceae, which comprises three species only, Humulus lupulus (the cultivated hop), H. japonicus and Cannabis sativa, all are dioecious and have evolved sex chromosomes of the X/autosome dosage type (Jacobsen, 1957; Parker, 1990). Silene latifolia and S. dioica, which form fully fertile hybrids and are clearly closely related, are the only two species with sex chromosomes in the genus Silene (of around 500 species) in the family Caryophyllaceae which contains in excess of 2000 species (Blackburn, 1923). In both species, males are XY and females XX. Although several species in the family Cucurbitaceae are dioecious, the presence of sex chromosomes has been authenticated only in Coccinia indica, which has an XX/XY system (Parker, 1990). In the family Loranthaceae (mistletoe), many Viscum species are dioecious and carry sex-speci®c chromosome rearrangements (Barlow and Martin, 1984). Phoenix dactylifera (date palm) is one of a number of dioecious members of the Palmae but is the only palm species with authenticated sex chromosomes, probably of the XX/XY type (Siljak-Yakovlev et al., 1996). The section Acetosa of the genus Rumex (Polygonaceae) contains about ten species characterized by the same X/autosome dosage system sex-chromosome system (Wilby and Parker, 1988). Females are XX whilst males are XY1Y2 (Kihara and Ono, 1923). In Rumex hastatulus, which evolved a sex chromosome system independently of R. acetosa, both X and Y chromosomes are involved in an intermediate system (Smith, 1972). Sex-chromosomes in ¯owering plants have evolved independently but have a number of features in common. The X and Y chromosomes are always the largest chromosomes in the complement (and probably in the entire genus) and the Y chromosomes (or summed Y-multiples) are much larger than the X in all species except Humulus lupulus and Viscum (Parker, 1990). Although it is clear that the evolution of plant sex chromosomes is associated with large increases in the DNA amount, the reasons behind these increases and the types of sequence are not well understood. In Rumex acetosa, sex chromosome speci®c repeated sequences have been described (Ruix Rejon et al., 1994; Shibata et al., 1999) and the sex chromosomes appear to contain large amounts of retroviral and viral related sequences. There is emerging evidence (Guttman and Charlesworth, 1998) that plant sex chromosomes show some sequence degeneracy, as is the case in animal Y chromosomes, where the non-pairing segment of the Y chromosome largely lacks functional loci. The application of the techniques of molecular biology will shed further light on the sex chromosomes of plants, the genes carried on them and the ways in which they have evolved. S E X D E T E R M I N AT I O N M E C H A N I S M S It is evident that the ¯oral dichotomy shown by dioecious plant species results from modi®cation during development of a perfect ¯ower by suppression of one or other organ sets. Comparison of male and female ¯owers from the various dioecious species reveals that the timing during ¯ower development of the suppression event is enormously variable between species. In a number of dioecious species, including Mercurialis annua (Durand and Durand, 1991), Cannabis sativa (Mohan Ram and Nath, 1964), Spinacia oleracea (Sherry et al., 1993), and Humulus species (Shephard, 1999), the divergence of the male and female developmental pathways occurs extremely early in ¯oral development and the inappropriate organs are not initiated; in all these species the male ¯owers resemble perfect ¯owers whilst the female ¯owers are strikingly dierent. In most species, however, both sets of sex organs are initiated and the inappropriate set of organs develops to some extent before abortion. This is the case in Silene latifolia (Grant et al., 1994a), Rumex AinsworthÐDioecy in Plants acetosa (Ainsworth et al., 1995) and Pistacia vera (Hormaza and Pollito, 1996). In a small number of dioecious species such as Actinidia deliciosa (Schmid, 1978) and Asparagus ocinalis (Galli et al., 1993; Caporali et al., 1994), the developmental divergence occurs so late that male and female ¯owers are super®cially indistinguishable from each other and from perfect ¯owers. In addition to timing dierences, those plants which initiate and arrest the inappropriate organ sets dier in the nature of this arrest. In some cases there is evidence of cell death (e.g. Asparagus ocinalis: Caporali et al., 1994; Actinidia deliciosa: Harvey and Fraser, 1988) whilst in others the cells of the arrested organ remain healthy (e.g. Rumex acetosa: Ainsworth et al., 1995; Silene latifolia: Farbos et al., 1997). In Silene latifolia, neither stamen nor pistil arrest occurs through cell death and involves dierentiation into parenchymatous cells in the case of the stamen in the female (Farbos et al., 1997). This variation in timing and nature of suppression argues for the existence of a variety of dierent underlying mechanisms. Despite increasing research eorts on a number of dierent plant species, there is relatively little information available on the molecular basis of sex determination and it is even dicult to estimate the numbers of genes involved, particularly as the genes which result in organ suppression are unlikely to be the primary sex determining genes. A possible exception is Ecballium elaterium where a single locus with three alleles determines sex (Mather, 1949). The two best studied systems are Silene latifolia and Rumex acetosa where sex is not labile and is underpinned by sex chromosome systems. Two main approaches have been adopted in attempts to isolate sex determining genes from plants: using homologues of genes known to be involved in ¯ower development in hermaphroditic model plants such as Arabidopsis or Antirrhinum, and using cloning strategies involving enrichment for sex chromosome sequences or enrichment for sex-linked transcripts. Homologues of genes involved in ¯ower development in hermaphroditic plants One of the ®rst molecular targets has been the MADS box genes which control the identities of the ¯oral organs in hermaphroditic plant species (reviewed by Weigel and Meyerowitz, 1994). A number of research groups working on dierent dioecious and monoecious species have reasoned that organ suppression might arise as a consequence of the dierential expression patterns of the B and C function genes. Mutants of the B function genes in hermaphroditic plants are eectively female with homeotic transformations of petals to sepals in the second whorl and stamens to carpels in the third whorl (Schwarz-Sommer et al., 1990). In Silene latifolia, it was found that the MADS box genes did not play a key role in sex determination and their expression patterns were much as predicted for model hermaphroditic plants with little dierence between male and female ¯owers (Hardenack et al., 1994). In cucumber, which is monoecious, the MADS box genes were also shown not to be associated with organ arrest (Perl-Treves et al., 1998). 215 In Rumex acetosa, the expression patterns of the putative B and C function homeotic genes were shown to be strikingly dierent from those seen in hermaphroditic species (Ainsworth et al., 1995). Of particular interest is that the C function gene showed a sex speci®c expression pattern. Gene expression is normal in male and female ¯owers, with expression in the stamen and carpel whorls, but expression is lost from the organs which cease to develop, the timing being coincident with the arrest of further organ growth. A similar situation has been found in Liquidambar styraci¯ua, a monoecious tree species, where C function expression was considerably reduced in the degenerating stamens of the male ¯ower (Liu et al., 1999). The key dierence between these two cases is that in Liquidambar the organ arrest is by cell death, whilst in Rumex the arrested cells appear healthy and other genes are expressed normally in the arrested organ (Ainsworth et al., 1995). Thus, whilst in Liquidambar the reduced C function expression is probably a consequence of cell death, the lack of C function expression in the arrested organs of Rumex may be a cause or a consequence of the arrest. Transgenic experiments are needed to resolve this question: constitutive expression of the C function in male or female plants should lead to the production of hermaphroditic ¯owers if the lack of the C function is the cause of organ arrest in male and female ¯owers. The involvement of organ identity genes in controlling the sex related dierences in organ development in unisexual species is more likely in those species which do not initiate the inappropriate organs and do not go through a hermaphrodite phase. The only such study has been on Humulus lupulus where clear sex related expression dierences in a putative B function gene were evident (Shephard, 1999). In Zea mays, a monoecious species, the Tasselseed (ts) loci cause the reversal of sex in tassel ¯orets, so that pistils develop and stamen development is suppressed (Emerson, 1920). The TASSELSEED2 gene has been cloned and encodes an alcohol dehydrogenase-like protein which has similarity to steroid dehydrogenases. Its function is necessary for gynoecium abortion of ¯owers in the tassel (DeLong et al., 1993). TASSELSEED2 homologues have also been isolated from Silene latifolia (STA1) and Arabidopsis thaliana (ATA1) (Lebel-Hardenack et al., 1997). However, expression of both genes is con®ned to the tapetal cells of the anther with no detectable expression in female tissues which argues for a conservation of function in Silene and Arabidopsis and also that STA1 is unlikely to function in sex determination in Silene (Lebel-Hardenack et al., 1997). This example illustrates the diculty associated with attempting to isolate sex determining genes based on homologues with known functions in other unisexual or hermaphroditic species. Hermaphroditic plants may well carry genes with sequences similar to sex determining genes but they are likely to have dierent functions in these species, the sex determining genes (as distinct from the downstream genes which result in the organ dierences) arising by gene duplication and functional divergence. 216 AinsworthÐDioecy in Plants Subtractive cloning A number of research groups have used subtraction techniques of either cDNA or genomic DNA in attempts to isolate sex determining genes from Silene latifolia. Dierential screening of a subtracted cDNA library enriched for male-speci®c sequences enabled nine Male Enhanced cDNA sequences (Men-1 to -10) to be isolated (Scutt et al., 1997b; Scutt and Gilmartin, 1998). Other research groups have also isolated some of these genes independently; MROS1 and MROS3 are homologous to Men-1 and Men-9, respectively (Matsunaga et al., 1996, 1997) and CCLS-4 is also homologous to Men-9 (Hinnisdaels et al., 1997). Of the Men genes, all but Men-9 were found to be expressed in the stamens during the earliest stages of male ¯ower development (although the duration of expression diered and also their response to the smut fungus, Ustilago violacea, which is able to induce the formation of stamens in genetically female ¯owers; Antonovics and Alexander, 1992). They are not expressed in female ¯owers. Men-6 diered from the others in that it was also expressed in petals from male ¯owers and is the ®rst example of a malespeci®c gene which is expressed in non-reproductive tissues of a dioecious plant. Men-9 was expressed strongly in male ¯ower buds, weakly in female buds and its expression was induced by the smut fungus. Men-9 is expressed in only the third (stamen) whorl of male and female ¯owers and delineates the third whorl itself (Robertson et al., 1997). Men-9 is also interesting in that it is probably the only Y chromosome located Men sequence (Guttmann and Charlesworth, 1998; Scutt et al., 1999). In terms of function, the Men genes appear to have diverse functions although a number may be involved in the synthesis of male-speci®c cell wall proteins (Scutt et al., 1999) and clearly more research is needed in this area. Subtraction using an asexual mutant of S. latifolia enabled Barbacar et al. (1997) to generate probes that were enriched for male sequences acting downstream of the sporogenous stage of male gametogenesis. Screening of a cDNA library yielded a number of clones of which 55 were studied in some detail and 22 were shown to be expressed during stamen development (Barbacar et al., 1997). Dierential screening of Silene latifolia cDNA libraries and subtraction techniques have mainly identi®ed genes involved in stamen development (i.e. the consequence rather than the cause of sex determination) and this is likely to be a continuing problem with this type of approach, given the abundance of the transcripts from the large number of genes involved in male gametogenesis. An alternative approach has been to focus on the S. latifolia Y chromosome by genomic subtraction. The sex chromosomes of dioecious plants are a good target for this type of approach because they are so large; in S. latifolia, sex chromosomes account for 16 % of the total DNA in the male genome (Matsunaga et al., 1994). The technique of representational dierence analysis (RDA), a method of genomic subtraction, was used by Donnison et al. (1996) to isolate male-speci®c DNA sequences. Using mutants carrying deletions of the Y chromosome, the positions of RDA markers on the Y chromosome were assessed, enabling linked genes for carpel suppression, stamen initiation and stamen maturation to be mapped. RDA mapping of much smaller Y chromosome deletions may ultimately allow these genes to be isolated (Donnison et al., 1996). Although not involved in sex determination, a gene (SIY1) located on the S. latifolia Y chromosome has recently been isolated by screening a cDNA library with a Y-speci®c probe generated by microdissection of the Y chromosome (Delichere et al., 1999). Microdissection of sex chromosomes is being used increasingly in attempts to isolate the genes involved in sex determination and to understand the nature of the sequences carried on the sex chromosomes (Buzek et al., 1997; Scutt et al., 1997a; Matsunaga et al., 1999; Shibata et al., 1999). Clearly, the search for the elusive primary sex determining gene will be extremely dicult and will be hampered by the sex dierentiation genes acting downstream. The approach most likely to be successful will probably involve analysis of the early events during the dierentiation of male and female in¯orescences and ¯owers by RNA ®ngerprinting. Dierential display (Liang and Pardee, 1992) allows the patterns of gene expression to be assessed during development of an organ or tissue but tends to generate a preponderance of 30 untranslated sequences and has generally not lived up to its initial promise. In Asparagus, for example, dierential display has proved unsuccessful in the isolation of sex-speci®c and developmental stage-speci®c sequences (Caporali et al., 1996; Marziani et al., 1999). However its successors, ¯uorescent dierential display (FDD; Scutt et al., 1999) and cDNAAFLP display (Bachem et al., 1996) are more robust and should enable transcripts which change in abundance during the key stages of ¯ower development (i.e. the window of sex determination) to be identi®ed and isolated. S TA B I L I T Y O F S E X S Y S T E M S Some plants, whether basically hermaphroditic, monoecious or dioecious, have extremely labile sex systems. These may be a result of an inability to control sex precisely in a complex environment (Korpelainen, 1998) or because lability confers an adaptive advantage (Charnov and Bull, 1973). In the latter case, the environment must exert some control over sex expression. Stress conditions and consequent resource limitations, such that hermaphrodites were unable to maintain both sex functions, might favour the separation of the sexes or might magnify the eects of inbreeding depression also favouring sex separation (Barrett, 1998). In terms of dioecy, labile systems include androdioecy, gynodioecy and subdioecy. For example, in Mercurialis annua, dioecious, monoecious and androdioecious populations are found in dierent parts of Europe and the local ecological conditions are important in determining the type of sex system (Pannell, 1997a, b). Analysis of the genetic basis of sex determination in M. annua implicates three unlinked loci, A, B1 and B2, with combinations of two alternative alleles at each locus (Louis, 1989; Durand and AinsworthÐDioecy in Plants Durand, 1991; Pannell, 1997a). These loci aect the levels of the plant hormones, auxins and cytokinins, which are found in the plants, and it is probable that sex in M. annua is brought about by modi®cation of the plant hormone biosynthetic pathways. Sex expression in Mercurialis is sensitive to exogenously applied hormones, auxins having a masculinizing eect and cytokinins a feminizing eect (Durand, 1969). Trans-zeatin, the speci®c cytokinin responsible for femaleness, accumulates to high levels in female shoot apices, but is undetectable as a free base in male shoot apices (trans-zeatin riboside accumulating instead) whilst auxin levels (IAA) are three to six times higher in male ¯owers than female ¯owers (Louis et al., 1990). Whatever the precise mechanism of sex determination in Mercurialis, it is clear that the auxin±cytokinin balance is important but it is not clear how the environment might interact with the three sex determining loci. Most dioecious species are less labile in sex expression than Mercurialis. In Rumex acetosa and Silene latifolia, the genetic basis of sex determination is strong, and there is little evidence for lability or environmental eects (Grant et al., 1994b). In R. acetosa and its dioecious relative R. acetosella, exogenously applied hormones are ineective in altering sexual development (Cula®c, 1999). This is also the case in Silene latifolia (Ye et al., 1991). By contrast, the stability of the Silene system is disrupted by infection with the anther smut fungus Ustilago violacea, where stamen development in chromosomally female plants can be induced with the anthers of infected plants containing U. violacea spores rather than pollen (Audran and Batcho, 1981). Infection of male plants can promote additional gynoecium development (Batcho and Audran, 1981). The causative agent provided by U. violacea infection remains unknown. In Humulus species and its relative Cannabis, despite the presence of X/autosome sex chromosome systems, sex determination appears to be somewhat leaky. In Humulus, monoecious phenotypes are not uncommon, particularly the development of terminal female ¯owers on in¯orescences of male plants (Shephard, 1999). In addition, male ¯owers can be induced to form on genetically female plants by the application of the weak synthetic auxin, alpha (2-chlorophenylthio) propionic acid (Weston, 1960). In Cannabis, auxins and ethylene have feminizing eects (Heslop-Harrison, 1956; Mohan Ram and Jaiswal, 1970) whereas cytokinins and gibberellins have masculinizing eects (Atal, 1959; Chailakhan, 1979). In some other dioecious plants, such as Maclura pomifera and Morus rubra, phytoestrogen levels have been implicated in sex determination (Maier et al., 1997). There seems to be little consensus on the eects of the various plant hormones on sex expression in plants, again arguing that the systems have evolved independently. M O L E C U L A R M A R K E R S FO R S E X Although molecular cloning approaches have not yet identi®ed primary sex determining genes in any dioecious plant species, a range of molecular markers linked to sex have been generated. These markers have either arisen from 217 genetic mapping programmes or from research aimed at ®nding sex-linked markers for agronomically important dioecious species. In dioecious plants cultivated for fruit or seed it is often dicult to identify females at an early stage of growth. Examples are kiwifruit (Actinidia deliciosa), hop (Humulus lupulus), date palm (Phoenix dactylifera), papaya (Carica papaya), pistachio (Pistacia vera), sea buckthorn (Hippophae rhamnoides) and cloudberry (Rubus chamaemorus). Many fewer male plants as pollen donors are generally required as compared with female plants. In sea buckthorn, for example, the ratio of females to males in cultivation is 1 : 9 (Persson and Nybom, 1998). In yams (Dioscorea species) grown for their edible tubers, the tuber yield from females is greater than from males (Akorodo et al., 1984). In a minority of dioecious plants, the males are agronomically superior to the females. Examples are long pepper (Piper longum, cultivated in India for its medical properties; Bannerjee et al., 1999), poplar (Populus species; Tschaplinski and Tuskan, 1994) and asparagus (Asparagus ocinalis; Benson, 1982), where the male plants are higher yielding than the females. The various molecular markers linked to sex include RAPDs, RFLPs, AFLPs and microsatellites. RAPD banding patterns have been linked to sex in Hippophae rhamnoides (Persson and Nybom, 1998), basket willow (Salix viminalis; Alstrom-Rapaport et al., 1998), Asparagus (Jiang and Sink, 1997), Piper longum (Bannerjee et al., 1999), Silene latifolia (Mulcahy et al., 1992; Di Stilio et al., 1998; Zhang et al., 1998), pistachio (Hormaza et al., 1994), cannabis (Cannabis sativa; Sakamoto et al., 1995), Actinidia chinensis (Harvey et al., 1997) and Atriplex garettii (Ruas et al., 1998). RFLP markers have been used to distinguish between the sexes in Asparagus (Bi et al., 1995). The increasing use of the AFLP technique has led to the identi®cation of sex-linked AFLPs in asparagus (Spada et al., 1998; Reamon-Buttner et al., 1999), Dioscorea tokoro (Terauchi and Kahl, 1999) and Rumex acetosa (Ainsworth et al., 1999). In Carica papaya, (GATA)n microsatellite banding patterns have been shown to be sex-speci®c (Parasnis et al., 1999). In Phoenix dactylifera, the sexes can readily be distinguished by cytological examination of interphase nuclei in root tip cells. Cells from male plants carry two ¯uorescent blocks of unequal intensity while female cells carry two equal blocks (Siljak-Yakovlev et al., 1996). In all cases but Salix viminalis and Actinidia, sex-linked markers have been linked to maleness. In cases where a sex chromosome system operates, it is clearly much more likely both that linkage will be found and that the linkage will be with maleness, as in all cases where the presence of sex chromosomes has been clearly established, the males are the heterogametic sex. In plants such as Silene latifolia, Cannabis sativa, Phoenix dactylifera and Rumex acetosa, therefore, it is not surprising that male-associated markers are relatively abundant. In dioecious plants where sex chromosomes have not been identi®ed, markers for maleness indicate either the presence of sex chromosomes which have not been distinguished by cytological methods or that the marker is tightly linked to a gene involved in sex determination. Based on the association of markers and 218 AinsworthÐDioecy in Plants sex, the presence of an XX/XY sex chromosome system has been claimed for Hippophae rhamnoides (Shchapov, 1979; Persson and Nybom, 1998), Dioscorea tokoro (Terauchi and Kahl, 1999), Carica papaya (Parasnis et al., 1999), Asparagus (Spada et al., 1998) and Actinidia (Harvey et al., 1997). By contrast, similar studies in Atriplex garettii suggest that sex determination involves a single locus on a homologous pair of chromosomes (Ruas et al., 1998). Female-associated molecular markers have been described in Actinidia (Harvey et al., 1997) and Salix viminalis (Alstrom-Rapaport et al., 1998). These might arise as a consequence of close linkage with a female sex determining gene or might indicate a sequence on the X chromosome inherited from the male parent. Salix viminalis is unlikely to have sex chromosomes and probably has a two locus epistatic system (Alstrom-Rapaport et al., 1998). CO N C L U S I O N S Eorts to understand the molecular basis of sex determination in dioecious plant species have, to date, not been successful. However, the developing molecular maps of several species (Dioscorea tokoro: Terauchi and Kahl, 1999; Asparagus: Spada et al., 1998) and RNA display methods seem likely to lead to the isolation of interesting genes in the future. Of particular interest in those cases of organ arrest where cell death is implicated, may be the genes which are involved in apoptosis. The addition of extracts from maturing male sex organs of Chara tomentosa to roots of Allium cepa or Melandrium nocti¯orum causes an almost complete arrest of cellular proliferation with cells arrested at G1 or G2 (Maszewski et al., 1998) suggesting that in dioecious plants where organ arrest occurs but without obvious cell death, such as in Rumex acetosa, the genes involved in the cell cycle will be worthy of study. When candidate genes are isolated, their roles in sex determination will need to be con®rmed in transgenic plants. Amongst the dioecious plant species, transformation protocols have been described only for Papaya (Mahon et al., 1996) and Humulus (Oriniakova and Matousek, 1996). Dioecious plants oer a unique opportunity for investigating plant development in a way which is complementary to the study of ¯ower development in hermaphroditic species. Indeed, we may learn a considerable amount about development in hermaphrodites from the study of unisexual plants. The genes controlling sex determination may not be recognized even if cloned from hermaphroditic plants such as arabidopsis. It is likely that sex determining genes may be present in the genomes of hermaphroditic plants, but may have quite dierent functions in some plants. Although the notion that dioecy may be controlled by simple mutations aecting male or female functions may hold true for some simple plant systems, the situation is clearly more complicated in the case of those dioecious plants which have developed sex chromosomes of the X to autosome dosage type, where dosage sensing mechanisms involving genes analogous to the numerator and denominator genes found in drosophila have developed. A distinction must also be made between the sex determining genes (and the genes involved in dosage sensing mechanisms) and those genes (the sex dierentiation genes) which result in the organ dierences between male and female ¯owers. 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