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Annals of Botany 86: 211±221, 2000
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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 di€erent 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 di€erent 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 di€erent 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 di€erent 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 e€ects 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 ocinalis)
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 di€erential 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 eciently.
Resource allocation can di€er 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
su€ering 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 di€erent 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
di€erent 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 a€ord 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).
Di€erentiated 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 di€erent. 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
ocinalis (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
di€erences, those plants which initiate and arrest the
inappropriate organ sets di€er in the nature of this arrest.
In some cases there is evidence of cell death (e.g. Asparagus
ocinalis: 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 di€erentiation 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 di€erent underlying
mechanisms.
Despite increasing research e€orts on a number of
di€erent plant species, there is relatively little information
available on the molecular basis of sex determination and it
is even dicult 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 di€erent dioecious and monoecious species have
reasoned that organ suppression might arise as a consequence of the di€erential expression patterns of the B and C
function genes. Mutants of the B function genes in
hermaphroditic plants are e€ectively 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 di€erence 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 di€erent 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 di€erence 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 di€erences 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 di€erences 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 diculty 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 di€erent functions in these
species, the sex determining genes (as distinct from the
downstream genes which result in the organ di€erences)
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. Di€erential 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
di€ered 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
di€ered 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). Di€erential 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 di€erence 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 dicult and will be hampered by
the sex di€erentiation genes acting downstream. The
approach most likely to be successful will probably involve
analysis of the early events during the di€erentiation of
male and female in¯orescences and ¯owers by RNA
®ngerprinting. Di€erential 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, di€erential 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
di€erential 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 e€ects 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 di€erent 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 a€ect 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 e€ect and cytokinins a feminizing e€ect
(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 e€ects (Grant
et al., 1994b). In R. acetosa and its dioecious relative
R. acetosella, exogenously applied hormones are ine€ective
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 e€ects
(Heslop-Harrison, 1956; Mohan Ram and Jaiswal, 1970)
whereas cytokinins and gibberellins have masculinizing
e€ects (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 e€ects 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 dicult 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
ocinalis; 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
E€orts 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 o€er 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 di€erent functions in some plants. Although the
notion that dioecy may be controlled by simple mutations
a€ecting 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 di€erentiation genes) which result in the organ
di€erences between male and female ¯owers. One ®nding
which may result from this work will be the way in which
genomes have evolved to reassign genes (or duplicate copies
of them) to new functions.
AC K N OW L E D G E M E N T S
The author is grateful to Dr Tracy McLellan for critical
reading of the manuscript.
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