Sex-Determining Mechanisms in Land Plants

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 reproducing 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 understanding 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” mechanism 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. 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. 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 archegoniophores (if female) that arise from the upper surface of the thallus (Figure 1). Antheridiophores produce sperm-forming antheridia, and archegoniophores produce egg-forming archegonia. 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 investigations. Working on the assumption that sex-determining factors exist on the Marchantia sex chromosomes, Okama and colleagues (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, indicating 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 understanding 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 representing ∼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 chromosomes in low copy number. Although all protein-encoding genes found within the human ampliconic sequences are expressed 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 chromosome 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 chromosome 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 are sequences in Marchantia as are in sequences may be necessary for gene the of repetitive sequences in the Marchantia Y chromosome and the relatively high of male-specific genes within these repetitive elements suggest gene conversion a in the repetitive elements of the Marchantia Y chromosome. important between Marchantia and is that occur in Marchantia all diploid are This that other are responsible for the of the X chromosome, although size may not be an of chromosome efforts to sequence the Y and X chromosomes in Marchantia will be very important in understanding how sex chromosomes sexual phenotype in this species and how both the X and Y chromosomes evolved in this ancient lineage of plants. group of land plants from an evolutionary is the which the and This lineage is most to the plants that on land to and Although the and of the are homosporous and produce only one of and are with their and that give to the female and male respectively. 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In male flowers form at the and female flowers form at the of each There are three major genes that the of unisexual flowers or their are the and genes the of and The gene is and the expression of the it to the of the of the The A gene is to and it is for the expression of The gene is for in that it is not for the of the the but for the of and in female and male to the and genes, plants are monoecious, plants are plants are and plants are male and In addition to the sex-determining genes, plant have long been in the sex-determining process in and and the of and can the of the with as a and as a and plants with of and or and and that is the of sex with of as a of them to a model for how sex determination might occur and with both as a of the female sex and an of the male The of the model are that the gene a that the and of production the to whereas the gene a that the and development This model is with how unisexual flowers might arise very and very the model also an of or in As by in the model of and and the of factors in the sex-determining process in account for the lack of results from several have molecular in of the theory of sex determination in Two genes, and have been identified in and one of them to the et al., The genome has only one copy whereas the genome has both The expression of both genes with sexual with plants or plants et al., et al., Although these studies are with the of not the of how in and not plants. et that the of the the of development by (i.e., to This that which is to be on the and the of males and females to which is to be on the are both important in sexual phenotype in cloning of the and genes will these to be Because sex determination in and most other species occurs of organs, et out to this is based on organ or within the The of homologs of the genes and the ability to them in these to that the sex determination in sex based on their their (i.e., in male are only in the and in female only in the In that develop in the of a are not et that gene might be of the sex-determining Even though this is a it the of studies have the of the genes in the sex determination process in many and plants, et al., et al., et al., 1999), et al., 2000), et al., Silene latifolia et al., and et al., In all the expression of or genes in or was to in for in the unisexual it is not the in expression of these genes are a or a of organ that sex-determining with genes is plants may not genes for sex determination not be that the unisexual flowers of most and plants are derived from flowers with all sex organ plant within the group of that has been is is a species with three and In this species, sexual phenotype is by a gene with three the the and the female) The of and females in a The lack of male that the is of a gene to the This and other to the that all males are females are the and males the flowers of different sexes also display sexual that with the The lack of between the responsible for and sex that both are and a sex chromosome although chromosomes not exist et al., The of has sex determination in this species, the from are by the by the female Because it is only that the sex of the individual can be molecular that with the have been To date, two have amplified DNA that are highly specific for males and but in females et al., 2002; et al., 2002). amplified DNA was also by DNA gel blot to be from the female genome et al., 2002), the molecular for between the Although is a and not a model plant it has a small genome of Mbp and and may be et al., with its in and regions of the make it is a species worthy of Silene latifolia is a species with individual plants either all female or all male flowers. As it is by the species to date, our of sex-determining mechanisms in plants will on this species. In male and female latifolia the and but development leading to unisexual flowers et al., The sexual phenotype of individuals is determined by sex males are and females are studies of sex-determining in latifolia that the Y chromosome is into three regions to sex one for the of female development and two for the of male of these regions be necessary for the development of female organs, these functions on the X or autosomal chromosomes. sex-determining have been recently by of and both and et al., 1999; et al., 1999; et al., 2002). the of were and have resulted in the of two classes of those that are not Y and and those that are Y and The are to a necessary for the whereas the the to identify or have not been Although these sex-determining genes have not been the of an amplified length of the Y chromosome for regions of the Y chromosome will be for genetic and physical of the sex-determining et al., and may to their to identify sex-determining genes in latifolia has been to genes that are expressed in the male flowers and their to the Y chromosome by Charlesworth, 2002). Of the genes that have been correlated with sex expression in latifolia to date, only the four in Sex in latifolia The Y homolog is and the X homolog is Sex in latifolia The Y homolog is and the X homolog is Although molecular have not yet in identifying the major sex-determining genes in this has and will to of how Y chromosomes evolved from an ancestral pair of autosomes in plants 2002; Negrutiu et al., that that in genetically determined males female genes are and females male genes are between the sex-determining genes be to or of is the that unisexual male and female will be in between chromosomes often is by the of on one homolog this the The lack of X-Y to and of gene on the Y chromosome, with the of genes for male and those necessary to female The latifolia Y chromosome not to this for several the Y chromosome is the X chromosome and is indicating that it may not be degenerate et al., and 2002). of DNA in gene have revealed that although the DNA of is that of the DNA of is that of et al., 2001; and 2002). that the latifolia sex chromosomes et al., Charlesworth, 2002), which is the to for human sex chromosome evolution and Page, 1999), it is that the latifolia Y chromosome is at a relatively of evolution 2002). sequencing of the latifolia sex chromosomes will be important in understanding the evolution of the Y chromosome in plants, especially with the Y chromosome of Marchantia and the sex-determining chromosome region of Sex determination in plants is a process that is important for the sexual of important how are and Although most plants are not model sex determination is not a that can be in the model in the sexual of plants to in this of studies of sex-determining mechanisms have that angiosperms, plants, have evolved a variety of sex-determining mechanisms that a number of different genetic and from sex chromosomes to plant Although the determinants of sexual phenotype are diverse, the genes that male or female development are in common or not will cloning the sex-determining genes from a variety of plant species. to sex determination in plants representing other major land plant lineages will several and evolutionary to be is how evolved from identifying the sex-determining genes in homosporous plants as Ceratopteris and the expression of possible genes in species, one can the that the from to a in the of expression of these genes from the gametophyte to the sporophyte Other to be are how sex chromosomes evolved in plants and similar processes to distinct sex chromosomes in plants and the Y chromosome sequences of Marchantia and for will be in understanding how genes and genes to a Y chromosome and how between the Y and its homolog was and to be was by the This is of the