The Plant Cell, Vol. 16, S61–S71, Supplement 2004, www.plantcell.
org ª 2004 American Society of Plant Biologists
Sex-Determining Mechanisms in Land Plants
Milos Tanurdzic and Jo Ann Banks1
Purdue Genetics Program and Department of Botany and Plant Pathology, Purdue University, West Lafayette, Indiana 47906
INTRODUCTION
Sex determination is a process that leads to the physical
separation of male and female gamete-producing structures to
different individuals of a species. Even though sexually repro-
ducing species have only three possible options—to relegate the
two sexes to separate individuals, to keep them together on
the same individual, or to have a combination of both—plants
in particular display a great variety of sexual phenotypes. In
angiosperms, a sex-determining process is manifest in species
that are monoecious, in which at least some flowers are
unisexual but the individual is not, or dioecious, in which
unisexual plants produce flowers of one sex type. In plants that
produce no flowers and are homosporous, sex determination is
manifest in the gametophyte generation with the production of
egg- and sperm-forming gametangia on separate individual
gametophytes. The determinants of sexual phenotype in plants
are diverse, ranging from sex chromosomes in Marchantia
polymorpha and Silene latifolia to hormonal regulation in Zea
mays and Cucumis sativa to pheromonal cross-talk between
individuals in Ceratopteris richardii. Here, we highlight recent
efforts aimed at understanding the genetic and molecular
mechanisms responsible for sex determination in several plant
species that separate their sexes into two individuals or flowers.
Representatives of all major land plant lineages are included to
give an evolutionary perspective, which is important in under-
standing how different sex-determining mechanisms evolved
and their consequences in plant development and evolution.
Although great progress has been made in genetically identifying
the genes that regulate sex expression in these species, few of
them have been cloned. Because a ‘‘one-size-fits-all’’ mecha-
nism of sex determination will not account for the variety of
sexual systems in plants, future efforts at cloning these genes
in several well-chosen model systems will be necessary to
understand these processes at the molecular level. There are
several excellent recent reviews of sex determination that
describe species that have not been included to which the
reader is directed (Ainsworth, 1999, 2000; Geber et al., 1999;
Matsunaga and Kawano, 2001; Negrutiu et al., 2001; Barrett,
2002; Charlesworth, 2002). We begin with the most basal lineage
of the land plants.
1 To
whom correspondence should be addressed. E-mail banksj@
purdue.edu; fax 765-494-5896.
Article, publication date, and citation information can be found at
www.plantcell.org/cgi/doi/10.1105/tpc.016667.
THE BRYOPHYTES
The bryophytes are a group of plants that includes the modern
liverworts, hornworts, and mosses. In this group, the haploid
gametophyte is the dominant phase of the life cycle, which is
illustrated in Figure 1. Their diploid spore-producing sporophytes
are very small, short-lived, and parasitic upon the independent
gamete-producing gametophyte generation. All bryophytes are
homosporous, producing only one type of spore, yet all three
groups of extant bryophytes are represented by species that are
homothallic, with one individual gametophyte producing both
male and female sex organs (gametangia), and species that are
heterothallic, with one individual producing only male or female
gametangia (Smith, 1955). Sexual dimorphism in heterothallic
species can be extreme, as exemplified by members of the
genus Micromitrium, in which the dwarf male gametophyte
grows on the leaves of the markedly larger female plant (Smith,
1955).
In many species of bryophytes, heterothallism (unisexuality)
has been correlated with the presence of sex chromosomes
(Smith, 1955). Although the extent of heterothallism and sex
chromosomes in the bryophytes has not been assessed
systematically, this is the only known group of homosporous
plants that uses sex chromosomes in sex determination. To
date, studies of bryophyte sex determination have focused on
the heterothallic liverwort Marchantia polymorpha. In this
species, the male and female thalli (vegetative gametophytes)
look alike, although males and females can be distinguished
easily by differences in the morphology of the sexual structure
each produces. A gametophyte bears gametangia on stalked
branches called antheridiophores (if male) or archegonio-
phores (if female) that arise from the upper surface of the
thallus (Figure 1). Antheridiophores produce sperm-forming
antheridia, and archegoniophores produce egg-forming arche-
gonia. The sex of each haploid gametophyte is determined by
cytologically distinct sex chromosomes, with males having one
very small Y chromosome and no X chromosome and females
having one X chromosome and no Y chromosome (Lorbeer,
1934). In addition to its rapid growth (it is often an invasive
weed in greenhouses), its ability to be propagated vegetatively
by gemma cups (Figure 1), and its ability to be transformed
(Takenaka et al., 2000), Marchantia has a relatively small
genome size of 280 Mbp distributed among eight autosomes
plus one sex chromosome (Okada et al., 2000), making it
a worthy model organism amenable to genomics-style inves-
tigations.
Working on the assumption that sex-determining factors exist
on the Marchantia sex chromosomes, Okama and colleagues
S62 The Plant Cell

Figure 1. The Life Cycle of the Liverwort Marchantia polymorpha.

Haploid gametophytes develop gametangia in antheridiophores and archegoniophores that produce the sperm and egg, respectively. Upon
fertilization, which is facilitated by raindrops, the diploid sporophyte remains attached to the archegoniophore and produces yellow sporangia, in which
haploid spores are formed after meiosis. The spores are liberated and germinate to form a new gametophyte thallus. The sex of the thallus depends on
which sex chromosome it inherits. Photographs courtesy of Katsuyuki Yamato, Kyoto University.

(2000) set out to identify these factors by constructing separate
male and female P1-derived artificial chromosome (PAC)
libraries and identifying clones specific to either the male or the
female genome. Their screen resulted in 70 male-specific PAC
clones that hybridized only the Y chromosome by fluorescence in
situ hybridization. No female-specific clones were found, in-
dicating that the X chromosome does not harbor long stretches
of unique sequences, as does the Y chromosome. To date, two
male-specific PAC clones with insert sizes totaling 126 kb have
been sequenced (Okada et al., 2001; Ishizaki et al., 2002). This
and other analyses have revealed that approximately one-fourth
to one-third of the 10-Mb Y chromosome of Marchantia consists
of an estimated 600 to 15,000 copies of an element of variable
length (0.7 to 5.2 kb) that contains other smaller repetitive
elements (Okada et al., 2001; Ishizaki et al., 2002). Of the six
putative protein-encoding genes found embedded within the
repeats, all are present in multiple copies on the Y chromosome
based on DNA gel blot hybridization. Two of these genes, named
ORF162 (Okada et al., 2001) and M2D3.5 (Ishizaki et al., 2002),
are unique to the Y chromosome; the remaining four genes are
present in low copy number on the X chromosome or the
autosomes. ORF162 encodes a putative protein with a RING
finger domain; M2D3.5 is a member of the same gene family.
ORF162 transcripts are detectable only in the male sexual
organs, indicating that the gene family represented by ORF162
and M2D3.5 may be important in the development of the
antheridiophore. Of the four genes also present on the X
chromosome or the autosomes, only one (M2D3.4) is restricted
in its expression to the male gametophyte, indicating that the
M2D3.4 X or automosmal homolog might be a pseudogene.
M2D3.4 encodes a putative protein similar to a Lilium longiflorum
gene that is expressed exclusively in the male gametic cells. The
remaining three genes are not sex specific in their expression.
The functions of the Y chromosome–encoded genes are as yet
unknown.
Although only a small portion of the Marchantia Y chromosome
has been sequenced, it is sufficient to make meaningful
comparisons with the euchromatic male-specific region (MSY)
of the human Y chromosome, the sequence of which was
published recently (Skaletsky et al., 2003; see also Hawley,
2003). The sequence of the human MSY region and limited
comparative sequencing of the MSY regions in great apes
(Rozen et al., 2003) have added new insights to our understand-
ing of how the mammalian testis gene families on the Y
chromosome have been maintained over the course of evolution.
As will be shown, the similarities between the human and
liverwort Y chromosomes are striking and may reflect a common
mechanism underlying the evolution of the Y chromosome in
these two disparate organisms. The MSY region of the human Y
chromosome is made up of three classes of sequences: X
transposed, X degenerate, and ampliconic, the latter represent-
ing
30% of the MSY euchromatin. Because the Marchantia X
chromosome has not been sequenced, it is only possible to
make comparisons between the human ampliconic sequences,
which are Y specific, and the Marchantia Y chromosome
sequences. Like the Marchantia Y chromosome sequences,
the ampliconic regions of the MSY consist of highly repetitive
sequences unique to the Y chromosome, although the sizes,
sequences, and stoichiometries of the repetitive elements very
considerably between the two species. Protein-encoding genes

or gene families (six genes in Marchantia and nine gene families
in human) occur within repetitive elements, and all are present
in multiple copies on the Y chromosome. In both organisms,
homologs also may be present on the X or autosomal chromo-
somes in low copy number. Although all protein-encoding
genes found within the human ampliconic sequences are ex-
pressed only in the testis, at least some of the protein-encoding
genes identified in Marchantia are male organ specific in their
expression.
According to the prevailing theory of mammalian sex chromo-
some evolution (Graves and Schmidt, 1992; Jegalian and Page,
1998; Lahn and Page, 1999), the X and Y chromosomes are
derived from an ancient autosomal pair of chromosomes. The Y
chromosome acquired genes, especially those that enhance
male fertility, by a series of autosomal transpositions that were
then amplified on the Y chromosome, whereas the X chromo-
some maintained its ancestral genes. Other genes present on the
Y chromosome (i.e., X homologs) were lost, probably aided by
a lack of X-Y recombination, leading to a mostly degenerate Y
chromosome. The recent sequencing results suggest that the
uniformity of the ampliconic repetitive sequences of the human Y
chromosome is maintained from generation to generation by
intrachromosomal Y-Y gene conversion. This occurs at relatively
high rates:
600 nucleotides per newborn human male are
estimated to have undergone Y-Y gene conversion (Rozen et al.,
2003). Although it is not known if there are higher order
palindromic repeated sequences in Marchantia as there are in
humans (these palindromic sequences may be necessary for
gene conversion), the homogeneity of repetitive sequences in the
Marchantia Y chromosome and the relatively high frequency of
male-specific genes within these repetitive elements suggest
gene conversion playing a role in maintaining the repetitive
elements of the Marchantia Y chromosome.
One important distinction between Marchantia and humans is
that X-X recombination cannot occur in Marchantia because all
diploid sporophytes are X-Y. This suggests that forces other than
homologous interchromosomal recombination are responsible
for preventing the degeneration of the X chromosome, although
size alone may not be an indicator of chromosome degeneracy.
Future efforts to sequence the entire Y and X chromosomes in
Marchantia will be very important in understanding how sex
chromosomes specify sexual phenotype in this species and how
both the X and Y chromosomes evolved in this ancient lineage of
plants.
THE LYCOPHYTES
Another group of land plants deserving attention from an
evolutionary perspective is the lycophyte lineage, which includes
the modern Lycopodiales genera Selaginella and Isoetes. This
lineage is most closely related to the earliest vascular plants that
first appeared on land
250 to 400 million years ago (Kenrick
and Crane, 1997). Although the earliest lycophytes and extant
members of the Lycopodiales are homosporous and produce
only one type of spore, Selaginella and Isoetes are heterospo-
rous, with their sporophytes producing free-living megaspores
and microspores that give rise to the female and male
gametophytes, respectively. Of the lycophytes, Selaginella has
Sex Determination S63
great potential as a useful comparative system for the study of
sex determination in plants, in part because many species have
very small genome sizes (J.A. Banks, unpublished observations).
Selaginella produces microsporangia and megasporangia that
are born on the same strobilus or in different strobili of the same
plant, depending on the species; strictly unisexual species have
not been reported. In Selaginella moellendorfii, for example, each
strobilis bears both kinds of sporangia, with microsporangia at
the bottom and macrosporangia toward the top of each strobilis.
In such plants, sex determination would be viewed as the
process that regulates the sexual identity of the sporangia in the
strobilis, a mechanism that has clear parallels with floral organ
identity in angiosperms that produce perfect flowers. Although
we know virtually nothing about this group of plants beyond
descriptive biology, this lineage holds important clues for
understanding the evolution of heterospory from homospory,
a switch that occurred many times during land plant evolution
(Stewart and Rothwell, 1993) and that has had a major impact on
the timing of sex determination from the gametophyte to
sporophyte generation in plants (Sussex, 1966). The question
of how heterospory evolved from homospory is difficult to study
in the heterosporous angiosperm lineage because their homo-
sporous progenitors are probably extinct.
THE HOMOSPOROUS FERNS
Recent phylogenetic analyses of vascular seed–free plants
group the leptosporangiate and eusporangiate ferns and
members of Equisetum and Psilotum into a monophyletic clade
that is sister to the seed plants (Pryer et al., 2001). With few
exceptions, they are homosporous plants. The one plant for
which a sex-determining pathway has been genetically well
defined is the leptosporangiate fern Ceratopteris richardii. Like
Marchantia, Ceratopteris is homosporous and produces only
one type of haploid spore. Although the sex of the Marchantia
gametophyte is determined genetically by sex chromosomes,
the sex of the Ceratopteris gametophyte (male or hermaphro-
ditic) is determined epigenetically by the pheromone antheridi-
ogen. Since their discovery by Dopp (1950) in the fern Pteridium
aquilinum, antheridiogens have been identified and character-
ized from many species of leptosporangiate ferns (reviewed by
Naf, 1979; Yamane, 1998), suggesting that it is a common mode
of regulating sexual phenotypes in this group of plants. Although
the structure of the Ceratopteris antheridiogen is unknown, all
other fern antheridiogens characterized to date are mostly novel
gibberellins (Yamane, 1998).
The Ceratopteris male and hermaphroditic gametophytes are
easy to distinguish not only by the type of gamete produced but
also by the presence or absence of a multicellular meristem. As
illustrated in Figure 2, the hermaphrodite forms a single meri-
stem and meristem notch that gives the hermaphrodite its
heart-shaped appearance. Cells of this meristem differentiate as
egg-forming archegonia, sperm-forming antheridia, or simply
enlarge, adding to the growing sheet of photosynthetic paren-
chyma cells that make up most of the hermaphrodite prothallus.
A Ceratopteris spore grown in isolation always develops as
a hermaphrodite. A male gametophyte develops from a spore
only if the spore is placed in medium that had previously
S64 The Plant Cell

Figure 2. The Sex-Determining Mutants of Ceratopteris richardii.

The her1 (hermaphroditic) mutant and the wild-type hermaphrodite are indistinguishable, as are the tra1 (transformer) mutant and the wild-type males,
except that the her1 and tra1 mutants are insensitive to the absence or presence of ACE. The ACE-insensitive fem1 (feminization) gametophyte produces
no antheridia. The man1 (many antheridia) mutant produces
10 times more antheridia than hermaphrodites, whereas the not1 (notchless) mutant
rarely produces antheridia. The meristem notch normally present on the hermaphrodite often is missing in the not1 mutant, giving it a cup-shaped
appearance. The novel phenotypes of the fem1 tra1 and fem1 not1 tra1 mutants are shown. an, antheridia; ar, archegonia; mn, meristem notch.

supported the growth of a hermaphrodite. Lacking a multicellular
meristem, almost all cells of the male gametophyte terminally
differentiate as antheridia. The male-inducing pheromone that
is secreted by the Ceratopteris hermaphrodite is called ACE for
antheridiogen Ceratopteris. Based on physiological studies in
Ceratopteris (Banks et al., 1993), ACE is not secreted by the
hermaphrodite until after it loses the competence to respond to
its male-inducing effects, which corresponds to the initiation of
the meristem. A gametophyte will develop as a male only if it is
exposed continuously to ACE from a very young age, between 2
to 4 days after spore inoculation. Thus, in a population of spores,
those that germinate first become ACE-secreting meristic
hermaphrodites, whereas those that germinate later become
ameristic males under the influence of ACE secreted by its
neighboring hermaphrodites.
To understand how ACE represses the development of fe-
male traits (i.e., archegonia and meristem) and promotes the
development of male traits (i.e., antheridia) in Ceratopteris,
a genetics approach has been used to identify the genes
involved in this response (Banks, 1998; Strain et al., 2001). To
date, five phenotypic classes of mutants have been identified;
representatives of each class are illustrated in Figure 2. In
addition to those that are always hermaphroditic (the hermaph-
roditic mutants), always male (the transformer [tra] mutants), or
always female (the feminization [fem] mutants) regardless of the
absence or presence of ACE, there are mutants that produce
excessive antheridia (the many antheridia mutants) as well as
the feminizing mutants that often lack a meristem notch (the
notchless mutants). By comparing the phenotypes of double
mutant gametophytes to each single mutant gametophyte
parent, the epistatic interactions among these genes have been
assessed. One particularly informative phenotype is the fem1
tra1 double mutant, also illustrated in Figure 2. Unlike other
double mutant combinations, this one has a novel phenotype
unlike that of either parent. This finding suggests that these two
genes (FEM1 and TRA1) define two separate pathways, one
specifying male development and the other female development.
The sex-determining mutants have been ordered into a genetic
sex-determining pathway, illustrated in Figure 3, that is most
consistent with the genetic data. In this pathway, the sex of the
gametophyte ultimately depends on the activities of two genes,
one specifying the development of male traits (FEM1) and the
other specifying the development of female traits (TRA). These
genes also repress each other, so that when TRA is active, FEM1
is not and visa versa. What determines which of these two genes
is expressed in the gametophyte (and thus its sex) is ACE, which
ultimately represses the TRA genes, as described in the legend
to Figure 3.
In comparing mechanisms of gametophytic sex determination
in homosporous bryophytes and ferns, one obvious question
that arises is what drove Marchantia to an X-Y chromosomal
mechanism of sex determination and Ceratopteris to an epi-
genetically regulated mechanism dependent on pheromonal
cross-talk between individuals? The answer to this question
probably lies in the different ratios of males and females or
hermaphrodites that occur in the populations of each species. In
Marchantia, the segregation of X and Y sex chromosomes during
meiosis in the sporophyte ensures that each gametophyte
progeny has an equal probability of being either male or female,
barring selection. In Ceratopteris, the ACE response allows
the ratio of males to hermaphrodites to vary depending on
the density of the population, such that as the population den-
sity increases, the proportion of males also increases. Although
the underlying sex-determining mechanism is inflexible in

Figure 3. The Genetic Sex-Determining Pathways in the Fern Ceratop-
teris, the Fly Drosophila melanogaster, and the Nematode Caenorhabdi-
tis elegans.
The genetic model of sex determination in Ceratopteris (Strain et al.,
2001) is dependent on two genes, FEM1 and TRA (there are at least two
TRA genes), which promote the differentiation of male (antheridia) and
female (meristem and archegonia) traits, respectively. FEM1 and TRA
also antagonize each other such that if FEM1 is active, TRA is not, and
vice versa. What determines which of these two genes prevails in the
gametophyte and thus its sex is the pheromone ACE, which activates the
HER genes, of which there are at least five, and sets into motion a series
of switches that ultimately result in male development (i.e., FEM1 on and
TRA off). These switches are thrown in the opposite direction when
spores germinate in the absence of ACE. Although FEM1 represses TRA
and TRA represses FEM1, they do not do so directly. TRA activates
MAN1, which represses FEM1, and FEM1 activates NOT1, which
represses TRA. Because TRA and FEM1 are the primary regulators of
sex, NOT1 and MAN1 are considered regulators of the regulators. The
sex determination pathway in C. elegans (Hodgkin, 1987; Villeneuve and
Meyer, 1990) is linear and consists of a series of negative control
switches. The state of the initial switch gene (xol-1) is dependent on the
ratio of X to autosomal (A) chromosomes. If the ultimate downstream
gene in this pathway (TRA-1) is high, the nematode develops as
a hermaphrodite. If TRA-1 is low, it develops as a male. The linear sex
determination pathway shown for Drosophila is from the mid-1980s
(Baker and Ridge, 1980; Cline, 1983). There are actually other sex-
determining pathways that account for most aspects of sexual
phenotype (reviewed by Oliver, 2002); the pathway shown is the somatic
pathway. In the soma, Sxl is the key regulator of sex, and its state of
activity is determined by the X:A ratio. The dsx gene is the downstream
regulatory gene that ultimately determines whether male or female genes
are expressed in the soma.
Marchantia, it is flexible enough in Ceratopteris to allow each
individual to determine its sex according to the size of the
population in which it resides and the speed at which it germi-
nates relative to its neighbors. The flexibility of the Ceratopteris
sex-determining mechanism is reflected in its sex-determining
pathway, and this becomes especially apparent compared with
the sex-determining pathways known from other organisms,
including Drosophila melanogaster and Caenorhabditis elegans,
which are illustrated in Figure 3. In both of these animals, an
individual’s sex (male or female in D. melanogaster and male or
hermaphrodite in C. elegans) is determined genetically by the
Sex Determination S65
ratio of X to autosomal chromosomes. This ratio is read and
either activates or represses the activities of downstream genes
in each pathway. In both animals, the sex ultimately depends on
the state of the terminal gene in each linear pathway, TRA1 in the
case of C. elegans. The Ceratopteris sex-determining pathway is
distinctly different from those of D. melanogaster and C. elegans
in that it is not linear and there are two sex-determining genes,
one for male and one for female development. Their ability to
repress each other endows each Ceratopteris spore with the
flexibility to determine its sex upon germination based on
environmental cues.
So why would a flexible mechanism of sex determination that
allows sex ratios to vary be adaptive in ferns but not in
bryophytes? The answer to this question may lie in the ephemeral
nature of the fern gametophyte. Although the gametophytes of
bryophytes are persistent, the gametophytes of ferns are not. In
Ceratopteris, for example, gametophytes reach sexual maturity
only 14 days after spore inoculation and die once they are
fertilized. The limited time that a fern gametophyte is able to be
crossed by another might favor a sex-determining system that
would promote outcrossing by increasing the proportion of
males when population densities are high and ensuring a high
proportion of hermaphrodites capable of self-fertilization when
population densities are low. Because there are a variety of sex-
determining mutants available in Ceratopteris, the hypothesis
related to the consequences of variable versus fixed sex ratios
can be tested easily, at least under defined laboratory conditions.
Future studies to clone the sex-determining genes in Cera-
topteris will be necessary to understand their biochemical
functions and to test their interactions predicted by the genetic
model. Although the size of the Ceratopteris genome is probably
very large (n ¼ 37), the ability to inactivate genes in the
Ceratopteris gametophyte by RNA interference (Stout et al.,
2003; G. Rutherford, M. Tanurdzic, and J.A. Banks, unpublished
observations) provides an important means to study the effects
of inactivating potential sex-determining genes in the Ceratop-
teris gametophyte.
THE FLOWERING PLANTS
Although unisexuality is very common in animals, hermaphro-
ditism is the rule in angiosperms. Approximately 90% of all
angiosperm species have perfect flowers with specialized
organs producing microspores or megaspores from which the
male or female gametophytes develop. Of the remaining species,
approximately half are monoecious, producing unisexual flowers
of both sexes on the same individual, and the other half are
dioecious, with unisexual male and female flowers arising on
separate individuals (Yampolsky and Yampolsky, 1922). The
distribution of dioecy and monoecy within the angiosperm
phylogenetic tree strongly favors the evolutionary scenario in
which unisexual flowers evolved from perfect flowers multiple
times in the angiosperm lineage (Lebel-Hardenack and Grant,
1997; Charlesworth, 2002). Not surprisingly, there are a variety of
sex determination mechanisms in the angiosperms. For organi-
zational purposes only, sex determination in monoecious and
dioecious species are treated separately in this review.
S66 The Plant Cell
The Monoecious Angiosperms
There are numerous terms to describe the variety of sexual
phenotypes observed in monoecious plant species (defined and
compiled by Sakai and Weller, 1999). Although this rich no-
menclature is appropriate, it tends to confound the problem
of sex determination in this group of plants. For simplicity,
monoecious species here are grouped into two categories: those
that produce only unisexual male and female flowers on the same
plant, and those that produce both unisexual and perfect flowers
on the same plant.
Zea mays (maize) is an example of a monoecious species that
produces only unisexual flowers in separate male and female
inflorescences, referred to as the tassel and ear, respectively.
Unisexuality in maize occurs through the selective elimination of
stamens in ear florets (flowers) and by the elimination of pistils in
tassel florets (reviewed by Irish, 1999). Two general classes of
sex-determining mutants have been identified in maize, including
those that masculinize ears and those that feminize tassels. The
anther ear (an1) and dwarf (d1, d2, d3, and d5) mutants of maize
are recessive and masculinize ears by preventing stamen
abortion in the female florets (Wu and Cheung, 2000). The
dominant dwarf mutation, d8, has a similar phenotype. The D1,
D3, and AN1 genes encode enzymes involved in the biosynthesis
of the plant hormone gibberellin (GA) (Bensen et al., 1995;
Winkler and Helentjaris, 1995; Spray et al., 1996). The D8 gene
encodes the maize homolog of GIBBERELLIN INSENSITIVE/
REPRESSOR OF gal-3 (Peng et al., 1999), a family of
transcription factors that negatively regulate GA responses in
plants (Richards et al., 2001; reviewed by Olszewski et al., 2002).
GA signaling is thought to be derepressed in the d8 mutant,
resulting in a dominant phenotype. The molecular identity of
these genes provides direct evidence that endogenous GAs
have a feminizing role in sex determination in maize.
The tasselseed1 (ts1) and ts2 mutants of maize feminize male
florets in the tassel, as illustrated in Figure 4. In addition to the
tassel phenotype, the second floret of the ear spikelet, which
normally fails to develop, develops normally in the ts mutants,
leading to a double-kerneled spikelet in the ear (Dellaporta and
Calderon-Urrea, 1994; Irish, 1999). The TS2 gene has been
cloned and shown to encode a putative short-chain alcohol
dehydrogenase with signature motifs of steroid dehydrogenases
(DeLong et al., 1993). TS2 mRNA is expressed in the sub-
epidermal layer of the gynoecium before its abortion, which
correlates well with the timing and location of cell death
responsible for the pistil abortion in wild-type male florets
(Calderon-Urrea and Dellaporta, 1999). The expression of ts2
requires the wild-type TS1 gene. Although the pistils of ear florets
also express TS1 and TS2, they are protected from a deathly fate
by the action of another gene, SILKLESS1, that interacts directly
or indirectly with TS2 (Calderon-Urrea and Dellaporta, 1999).
Although the cloning of the sex-determining genes in maize
demonstrates that GAs and possibly other steroid-like hormones
play a pivotal role in stamen abortion and feminization of flowers,
the spatial distribution of these molecules could have an effect on
the sex determination process, as exemplified by a steep
gradient in GA abundance along the maize shoot (Rood et al.,
1980), which correlates well with the male-suppressing and
Figure 4. The ts4 Mutant of Maize.
The wild-type male tassel (left) produces only male florets, whereas
plants homozygous for the ts4 allele (right) form hermaphroditic flowers
with functional pistils, allowing seeds to form in the tassel. Photographs
courtesy of Erin Irish, University of Iowa.
female-promoting phenotypic effects of GA. How the synthesis
or transport of these molecules is regulated, and the identity
of the downstream targets of these hormones, remain to be
discovered.
Another monoecious plant, Cucumis sativus (cucumber), has
served as a model system for sex determination studies since the
1950s and early 1960s, driven by breeding programs for hybrid
seed production. Cucumber plants are mostly monoecious but
can be dioecious or hermaphroditic, depending on the genotype.
Regardless of their sex, all floral buds are initially hermaphroditic,
and it is the arrest of stamen or pistil development that leads to
unisexual flowers. In monoecious cucumbers, male flowers form
at the bottom and female flowers form at the top of each shoot.
There are three major genes that affect the arrangement of
unisexual flowers or their sex; they are designated the F, A, and M
genes (following the nomenclature of Pierce and Werner, 1990).
The F gene is semidominant and affects the expression of
femaleness along the plant, causing it to extend the gradient
of femaleness toward the bottom of the plant. The A gene
is epistatic to F, and it too is required for the expression of
femaleness. The M gene is required for maleness in that it is not
required for the establishment of the gender gradient along the
shoot but rather for the selective abortion of pistils and stamens
in female and male flowers, respectively (Perl-Treves, 1999).
With respect to the F and M genes, M-ff plants are monoecious,
M-F- plants are female, mmF- plants are hermaphroditic, and
mmff plants are andromonoecious (with male and hermaphro-
ditic flowers).
In addition to the sex-determining genes, plant hormones have
long been implicated in the sex-determining process in cucum-
ber. GA and ethylene application and the use of GA and ethylene
inhibitors can subvert the genotypic constitution of the plant, with
GA acting mainly as a masculinizing agent and ethylene acting
as a feminizing agent (Perl-Treves, 1999). By treating monoe-
cious and andromonoecious cucumber plants with various

combinations of GA and ethrel or GA and ethylene inhibitors, Yin
and Quinn (1995) demonstrated that ethylene is the main reg-
ulator of sex determination, with GA functioning upstream of
ethylene, possibly as a negative regulator of endogenous
ethylene production. These findings led them to propose a model
for how sex determination might occur (Yin and Quinn, 1995),
with ethylene serving both as a promoter of the female sex and an
inhibitor of the male sex. The basic tenets of the model are that
the F gene should encode a molecule that would determine the
range and gradient of ethylene production along the shoot,
thereby acting to promote femaleness, whereas the M gene
should encode a molecule that perceives the ethylene signal and
inhibits stamen development above threshold ethylene levels.
This model is consistent with how unisexual flowers might arise
very early and very late during shoot development; however, the
model also predicts an entire range of intermediate types rarely
or never seen in cucumber. As suggested by Perl-Treves (1999),
variations in the model of Yin and Quinn and the incorporation of
additional factors in the sex-determining process in cucumber
could account for the observed lack of intermediate types.
Recent results from several laboratories have provided
molecular evidence in favor of the ethylene theory of sex
determination in cucumber. Two 1-aminocyclopropane-1-car-
boxylic acid synthase genes, CS-ACS1 and CS-ACS2, have
been identified in cucumber, and one of them (CS-ACS1) maps
to the F locus (Trebitsh et al., 1997). The monoecious cucumber
genome has only one copy (Cs-ACS1), whereas the gynoecious
genome has both copies. The expression of both genes
correlates with sexual phenotype, with gynoecious plants
accumulating more transcript than monoecious or andromonoe-
cious plants (Kamachi et al., 1997; Yamasaki et al., 2001).
Although these studies are consistent with the female-promot-
ing effects of ethylene, they do not address the question of
how ethylene inhibits stamen abortion in gynoecious and not
andromonoecious plants. Yamasaki et al. (2001) provided
evidence suggesting that the product of the M locus mediates
the inhibition of stamen development by ethylene (i.e., M affects
sensitivity to ethylene). This finding indicates that ethylene
concentration, which is likely to be dependent on the F locus,
and the differential sensitivity of males and females to ethylene,
which is likely to be dependent on the M locus, are both
important in regulating sexual phenotype in cucumber. Definitive
cloning of the M and F genes will allow these hypotheses to be
tested directly.
Because sex determination in cucumber and most other
angiosperm species occurs via selective abortion of flower
organs, Kater et al. (2001) set out to establish whether this
abortion is based on organ identity or positional information
within the flower. The availability of cucumber homologs of the
MADS box ABC homeotic genes and the ability to express them
ectopically in cucumber allowed these authors to show that the
sex determination machinery in cucumber selectively aborts sex
organs based on their position rather than their identity (i.e., in
male flowers, carpels are aborted only in the fourth whorl, and in
female flowers, stamens abort only in the third whorl). In addition,
because nonreproductive organs that develop in the inner whorls
of a C-class homeotic mutant are not aborted, Kater et al. (2001)
speculated that C-class gene products might be targets of the
Sex Determination S67
sex-determining process. Even though this is a tempting
speculation, it leaves the question of abortion timing unresolved.
Several studies have addressed the role of the MADS box floral
homeotic genes in the sex determination process in many mono-
ecious and dioecious plants, including Asparagus officinalis
(Park et al., 2003), Betula pendula (Elo et al., 2001), Gerbera
hybrida (Yu et al., 1999), Populus deltoides (Sheppard et al.,
2000), Rumex acetosa (Ainsworth et al., 1995), Silene latifolia
(Hardenack et al., 1994), and maize (Heuer et al., 2001). In all
cases, the expression of B- or C-function floral homeotic genes
in carpels or stamens was shown to decline in organs targeted
for abortion in the unisexual flower. However, it is not clear if
the changes in expression of these genes are a cause or a con-
sequence of organ abortion. Furthermore, evidence that sex-
determining mutants cosegregate with MADS box genes is
lacking. That plants may not use MADS box genes for sex
determination would not be surprising given that the unisexual
flowers of most monoecious and dioecious plants are derived
from bisexual flowers with all sex organ primordia present.
Another plant within the monoecious group of angiosperms
that has been studied is Carica papaya (papaya). Papaya is
a polygamous species with three sexes—females, males, and
hermaphrodites. In this species, sexual phenotype is controlled
by a single gene with three alleles: the dominant M (for male), the
dominant Mh (for hermaphrodite), and the recessive m (for
female) alleles (Storey, 1938). The progeny of self-fertilized
hermaphrodites (genotypically Mhm) segregate hermaphrodites
and females in a 2:1 ratio. The lack of male progeny indicate that
the MM genotype is lethal, perhaps because of a lethal gene
closely linked to the M locus. This and other crosses led early
workers to the conclusion that all males are genotypically MhM.
Thus, females are the homogametic and males the hetero-
gametic sex. Papaya flowers of different sexes also display
secondary sexual characteristics that cosegregate with the M
allele. The apparent lack of recombination between the loci
responsible for primary and secondary sex traits indicates that
both loci are tightly linked and inherited en bloc, much like a sex
chromosome (Storey, 1953), although heteromorphic chromo-
somes do not exist (Kumar et al., 1945).
The economical importance of papaya has driven sex de-
termination research in this species, because the pyriform fruits
from hermaphroditic trees are preferred by consumers more than
the spherical fruits produced by the female trees. Because it is
only upon flowering that the sex of the individual papaya tree can
be determined, molecular markers that cosegregate with the M
alleles have been sought intensively. To date, two groups have
reported randomly amplified polymorphic DNA markers that are
highly specific for males and hermaphrodites but absent in
females (Deputy et al., 2002; Urasaki et al., 2002). One randomly
amplified polymorphic DNA marker was also shown by DNA gel
blot hybridization to be completely absent from the female ge-
nome (Urasaki et al., 2002), providing the first molecular evidence
for genomic differentiation between the sexes. Although papaya
is a tropical tree and not considered a model plant system, it has
a small genome of 371 Mbp (Arumuganathan and Earle, 1991)
and may be transformable (Fitch et al., 1992). These facts,
coupled with its economic importance in tropical and subtropical
regions of the world, make it is a species worthy of further study.
S68 The Plant Cell
The Dioecious Angiosperms
Silene latifolia is a dioecious species with individual plants
producing either all female or all male flowers. As it is by far the
best characterized dioecious species to date, our review of sex-
determining mechanisms in dioecious plants will focus on this
species. In male and female S. latifolia flowers, the gynoecium
and androecium initiate but arrest development prematurely,
leading to functionally unisexual flowers (Grant et al., 1994). The
sexual phenotype of individuals is determined by sex chromo-
somes; males are heterogametic (XY) and females are homoga-
metic (XX). Early cytogenetic studies of sex-determining mutants
in S. latifolia led Westergaard (1946, 1958) to conclude that the
Y chromosome is divided into three regions relevant to sex
expression: one required for the suppression of female de-
velopment and two required for the promotion of male de-
velopment. None of these regions would be necessary for the
development of female reproductive organs, because these
functions would reside on the X or autosomal chromosomes.
Additional sex-determining mutants have been generated re-
cently by x-ray mutagenesis of pollen and selecting both
hermaphrodites and asexual F1 progeny (Farbos et al., 1999;
Lardon et al., 1999; Lebel-Hardenack et al., 2002). These
mutants verify the earlier work of Westergaard (1946, 1958; his
lines were apparently lost) and have resulted in the identification
of two additional classes of mutants, those that are not Y linked
and hermaphroditic and those that are Y linked and asexual. The
hermaphroditic deletion mutants are likely to contain a gene(s)
necessary for the female-suppressing function, whereas the
asexual deletion mutants likely contain the male-promoting
gene(s). Genetic screens to identify mutant XX hermaphrodites
or asexuals have not been reported. Although these sex-
determining genes have not been cloned, the construction of
an amplified fragment length polymorphism map of the Y
chromosome using lines deleted for overlapping regions of the
Y chromosome will be useful for genetic and physical mapping of
the sex-determining mutants (Lebel-Hardenack et al., 2002) and
may ultimately lead to their cloning.
Another approach to identify sex-determining genes in S.
latifolia has been to clone genes that are expressed specifically in
the male flowers and determine their linkage to the Y chromo-
some (reviewed by Charlesworth, 2002). Of the >50 isolated
genes that have been correlated with sex expression in S. latifolia
to date, only the four listed in Table 1 have been shown to be
linked to the Y chromosome. Genes linked exclusively to the Y
chromosome have not been found, because all of the Y-linked
Table 1. Sex Chromosome–Linked Genes in S. latifolia
Y-Linked Gene X-Linked Gene Male-Specific Expression
SlY-1 SlX-1 SlY-1 yes, SlX-1 no
SlY-4 SlX-4 No
MRO3-Ya MRO3-Xa Yes
DD44Y DD44X No
a The Y homolog is inactive and the X homolog is active.
genes have X or autosomal homologs. These data indicate
that the S. latifolia Y chromosome most closely resembles the
X-degenerate class of sequences of the human Y chromo-
some. Genes within this class have an X homolog, the X and
Y homologs encode similar but nonidentical isoforms, many of
them are ubiquitously expressed, and all are present in low copy
number in the genome (Skaletsky et al., 2003). As shown in Table
1, many of these features are shared with the Y-linked genes of
S. latifolia.
Although molecular approaches have not yet succeeded in
identifying the major regulatory sex-determining genes in S.
latifolia, this work has and will continue to test theories of how Y
chromosomes evolved from an ancestral pair of autosomes in
plants (Charlesworth, 1996, 2002; Negrutiu et al., 2001). These
theories state that once mutations that result in genetically
determined males (where female genes are repressed) and
females (where male genes are repressed) occur, recombination
between the sex-determining genes must be suppressed to
avoid recombinant asexual or hermaphroditic offspring. Another
consequence of nonrecombination is the certainty that unisexual
male and female offspring will be produced in equal ratios.
Recombination between homologous chromosomes often is
suppressed by the accumulation of chromosomal inversions on
one homolog (in this case, the Y). The lack of X-Y recombination
would eventually lead to degeneracy and loss of gene function on
the Y chromosome, with the exception of genes required for male
fertility and those necessary to suppress female fertility. The S.
latifolia Y chromosome appears not to fit this paradigm for
several reasons. First, the Y chromosome is 1.4 times larger than
the X chromosome and is largely euchromatic, indicating that it
may not be degenerate (Ciupercescu et al., 1990; Grabowska-
Joachimiak and Joachimiak, 2002). Second, measurements of
DNA polymorphism in sex-linked gene pairs have revealed that
although the DNA polymorphism of S4Y-1 is greater than that of
S4X-1, the DNA polymorphism of SlY-1 is 20-fold lower than that
of SlX-1 (Filatov et al., 2000, 2001; Filatov and Charlesworth
2002). Given that the S. latifolia sex chromosomes diverged less
than
20 million years ago (Desfeux et al., 1996; Charlesworth,
2002), which is much less than the 240- to 320-million-year
timeline for human sex chromosome evolution (Lahn and Page,
1999), it is likely that the S. latifolia Y chromosome is at a relatively
young stage of evolution (Charlesworth, 2002). Comparative
sequencing of the S. latifolia sex chromosomes will be important
in understanding the evolution of the Y chromosome in plants,
especially compared with the Y chromosome of Marchantia and
the sex-determining chromosome region of papaya.
Function Reference
WD-repeat protein Delichere et al., 1999
Fructose-2,6-bisphosphatase Atanassov et al., 2001
Unknown Matsunaga et al., 1996; Guttman and
Charlesworth 1998
Oligomycin sensitivity–conferring Moore et al., 2003
protein

FUTURE DIRECTIONS
Sex determination in plants is a fundamental developmental
process that is particularly important for economic reasons,
because the sexual phenotypes of commercially important crops
dictate how they are bred and cultivated. Although most crop
plants are not considered model systems—and sex determina-
tion is not a problem that can be addressed in the model
angiosperm Arabidopsis—the economic value in manipulat-
ing the sexual phenotypes of crop plants should continue to
drive interest in this area of research. Recent studies of sex-
determining mechanisms have demonstrated clearly that an-
giosperms, including crop plants, have evolved a variety of
sex-determining mechanisms that involve a number of different
genetic and epigenetic factors, from sex chromosomes to plant
hormones. Although the determinants of sexual phenotype are
diverse, determining whether the downstream master sex-
regulatory genes that specify male or female development are
held in common or not will require cloning the sex-determining
genes from a variety of plant species.
Choosing to study sex determination in plants representing
other major land plant lineages will allow several broader
developmental and evolutionary questions to be addressed.
One unresolved question is how heterospory evolved from
homospory. By identifying the sex-determining genes in homo-
sporous plants such as Ceratopteris and examining the
expression of possible homologous genes in closely related
heterosporous species, one can test the hypothesis that the
switch from homospory to heterospory involved a heterochronic
shift in the timing of expression of these genes from the
gametophyte to the sporophyte generation. Other questions to
be resolved are how sex chromosomes evolved in plants and
whether similar processes led to distinct sex chromosomes in
plants and animals. Comparing the Y chromosome sequences of
Marchantia and S. latifolia, for example, will be invaluable in
understanding how male-promoting genes and female-sup-
pressing genes became localized to a Y chromosome and how
recombination between the Y and its homolog was and con-
tinues to be suppressed.
ACKNOWLEDGMENTS
Support was provided by the National Science Foundation (MCB-
9723154). This is journal paper 17271 of the Purdue University
Agricultural Experiment Station.
Received August 25, 2003; accepted January 19, 2004.
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