- Email: [email protected]
Molecular and evolutionary analysis of a plant Y chromosome
C.R. Acad. Sci. Paris, Sciences de la vie / Life Sciences 324 (2001) 531–535 © 2001 Académie des sciences/Éditions scientifiques et médicales Elsevier SAS. Tous droits réservés S0764446901013221/FLA
Review / Revue
Molecular and evolutionary analysis of a plant Y chromosome Françoise Monéger* Laboratoire de reproduction et développement des plantes, ENS Lyon, 46, allée d’Italie, 69364 Lyon cedex 07, France Received 23 October 2000; accepted 4 December 2000 Communicated by Christan Dumas
Abstract – Plants have evolved a great diversity of sex determination systems. Among these, the XY system, also found in mammals, is one of the most exciting since it gives the opportunity to compare the evolution of sex chromosomes in two different kingdoms. Whereas genetic and molecular mechanisms controlling sex determination in drosophila and mammals, have been well studied, very little is known about such processes in plants. White campion (Silene latifolia) is an example of plant with X and Y chromosomes. What is the origin of the X and Y chromosomes? How did they evolve from a pair of autosomes? In our laboratory, we have isolated the first active genes located on a plant Y chromosome. We are using them as markers to trace the origin and evolution of sex chromosomes in the Silene genus. © 2001 Académie des sciences/Éditions scientifiques et médicales Elsevier SAS sex chromosomes / Silene latifolia / molecular evolution / Y-linked genes / sex determination
Résumé – Analyse moléculaire et évolutive d’un chromosome Y végétal. Les plantes montrent une grande diversité dans leurs systèmes de détermination du sexe. Parmi eux, celui basé sur la présence de chromosomes XY semblables à ceux des mammifères est le plus attractif car il ouvre la possibilité de comparer l’évolution des chromosomes sexuels dans les deux règnes. Tandis que les mécanismes génétiques et moléculaires contrôlant la détermination du sexe ont été bien étudiés chez la drosophile et les mammifères, très peu de choses sont connues chez les plantes. Le compagnon blanc (Silene latifolia) est un exemple de plante modèle avec des chromosomes X et Y. Quelle est l’origine des chromosomes X et Y ? Comment ont-ils évolué à partir d’une paire d’autosomes ? Dans notre laboratoire, nous avons isolé les premiers gènes actifs localisés sur un chromosome Y végétal. Nous les utilisons aussi comme marqueurs pour retracer l’origine et l’évolution des chromosomes sexuels dans le genre Silene. © 2001 Académie des sciences/Éditions scientifiques et médicales Elsevier SAS chromosomes sexuels / Silene latifolia / évolution moléculaire / gènes liés au Y / détermination du sexe
. Version abrégée Les plantes montrent une grande diversité dans leurs systèmes de détermination du sexe. Parmi eux, celui basé sur la présence de chromosomes XY semblables à ceux des mammifères est le plus attractif car il ouvre la possibilité de comparer l’évolution des chromosomes
sexuels dans les deux règnes. Tandis que les mécanismes génétiques et moléculaires contrôlant la détermination du sexe chez la drosophile et les mammifères ont été bien étudiés, très peu de choses sont connues sur de tels processus chez les plantes. Le compagnon blanc (Silene latifolia) est un exemple de plante avec des chromosomes X et Y. À un stade très précoce, les
*Correspondence and reprints. E-mail address: [email protected] (F. Monéger).
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F. Monéger / C.R. Acad. Sci. Paris, Sciences de la vie / Life Sciences 324 (2001) 531–535
méristèmes floraux du compagnon blanc renferment à la fois les primordia d’organes mâles et femelles. Chez les plantes mâles, la présence d’un chromosome Y fonctionnel : 1) inhibe la prolifération cellulaire dans le territoire femelle, et 2) active le développement des organes mâles. Chez les plantes femelles (XX), le développement des organes femelles se déroule normalement (en l’absence d’inhibition), et l’absence d’activation mâle résulte dans l’arrêt du développement des organes mâles. Le chromosome Y porte aussi des gènes impliqués dans la fertilité mâle. Enfin, les chromosomes X et Y partagent une région pseudoautosomale (PAR) par laquelle ils s’apparient pendant la méiose. Quelle est l’origine des chromosomes X et Y ? Comment ont-ils évolué à partir d’une paire d’autosomes ? Dans notre laboratoire, nous avons isolé les premiers gènes actifs localisés sur un chromosome Y végétal. L’un d’eux (SlY1 pour ‘Silene latifolia gène
Y1’) code une protéine qui contient des domaines ‘WD-repeat’ et qui est vraisemblablement impliquée dans la prolifération cellulaire. Ce gène a un homologue sur le chromosome X (SlX1). Les deux gènes présentent un profil d’expression similaire, mis à part que SlY1 est absent des plantes femelles. La comparaison de séquences entre SlY1 et SlX1 nous permet d’estimer à quel moment les deux gènes ont cessé de recombiner, soit il y a environ 2,5 millions d’années. Ce genre d’analyse conduit sur plusieurs loci devrait nous permettre de dater l’émergence des chromosomes sexuels. Les cinq gènes que nous avons identifiés sur les chromosomes X et Y vont nous servir de marqueurs pour retracer l’histoire des chromosomes sexuels dans le genre Silene. Les chromosomes XY de plantes ont évolué beaucoup plus récemment que ceux des mammifères et nous donnent donc accès aux étapes initiales de l’évolution des chromosomes sexuels.
1. Introduction
cesses in plants. We have chosen to analyse sex determination in white campion (Silene latifolia) which is one of the most suitable plant species in which to address such questions. Sex determination in this Silene was reported, as in mammals, to be under the strict genetic control of a dominant Y chromosome [8, 9]. XX individuals develop female flowers, whereas XY individuals develop male flowers. The presence or absence of the Y chromosome determines the sex.
Most of the flowering plants are hermaphrodite. However, separation of sexes has been observed for a long time and was already mentioned in the 1889 book entitled The evolution of sex by P. Geddes and A. Thomson. At the beginning of the twentieth century, De Vries, Correns, Bateson, Baur, Shull and Winge made more or less extensive investigations in Silene latifolia : the presence of sex chromosomes in a plant , white campion (Silene latifolia, also called Melandrium album) was reported. In 1948, Westergaard demonstrated the active nature of the Y chromosome in white campion, by showing that a single Y chromosome is sufficient to induce the development of male flowers, even in the presence of several X chromosomes [1]. It is striking to note the ressemblance of this sex determination system to that of mammals. This convergent evolution of sex determination mechanisms raises exciting and challenging questions about structure, function and evolution of sex chromosomes in the two kingdoms. The majority of flowering plants (72 %) possess hermaphrodite flowers. However, different forms of sexuality exist : monoecy (separate unisexual flowers on the same plant, as in Zea mays) occurs in 7 % of higher plants; intermediates (both hermaphrodite and unisexual flowers on the same planta, as in Silene otites) which represent 17 %; and finally dioecy (unisexual flowers on separate plants, as in Asparagus officinalis) which occurs in 4 %. Some of the dioecious species have heteromorphic sex chromosomes. In animals, sex determination systems can be based on the X/autosome ratio (as in drosophila), or on X/Y with a dominant Y chromosome (as in mammals). Both systems exist in the plant kingdom [2, 3]. While genetic and molecular mechanisms controlling sex determination in drosophila and mammals have been extensively studied [4–7], very little is known about such pro-
532
At a very early stage, floral meristems from both sexes are potentially hermaphrodite : they contain both male and female reproductive organ primordia (figure 1). The first function on the Y chromosome to operate in male flowers is what we have called GSF (for gynoecium suppressing function). GSF is responsible for the sudden arrest of cell proliferation in the center of the flower meristem, which would otherwise develop into a gynoecium (female organ). The second function on the Y chromosome promotes stamen (male organ) development. As a result, in a male XY plant, flowers will develop with normal stamens and an undifferentiated filamentous structure instead of the gynoecium in the center of the flower (figure 1). Conversely, in a female XX plant, flowers will develop with a normal gynoecium but stamens will stop developing at an early stage [10]. The Y chromosome also carries male fertility genes (figure 1). The presence of these functions as well as their location on the Y chromosome, has been deduced from the sexual phenotype of mutants carrying Y chromosome deletions induced by gamma-irradiation. Finally, the X and Y chromosomes share a pseudoautosomal region (PAR) through which they specifically pair during meiosis [9, 11]. To date, our knowledge of the Y chromosome is limited to morphological, genetic and cytogenetic data. Several questions still remain to be answered. Does the Y chromosome from Silene latifolia, contain housekeeping genes as has been shown to be the
F. Monéger / C.R. Acad. Sci. Paris, Sciences de la vie / Life Sciences 324 (2001) 531–535
chromosomes on metaphase spreads from root cultures and used them in DOP-PCR (degenerated oligonucleotide primer–polymerase chain reaction) amplification. We used this Y-specific DNA to probe a cDNA library constructed from male flower buds. Among the positive clones, we isolated five cDNAs which hybridize to male-specific restriction fragments. These genes represent the first expressed genes identified on a plant Y chromosome. One of these genes, SlY1 (for Silene Latifolia Y1 gene), has been analysed in detail [13]. SlY1 is expressed predominantly in male flowers. A closely related gene, SlX1, is located on the X chromosome and is strongly expressed in both male and female flowers. SlY1 and SlX1 encode almost identical proteins containing WD-repeats. Such proteins are found almost exclusively in eukaryotes, where they are involved in a variety of processes including regulation of transcription, control of cell growth and differentiation, and chromatin structure [14–17]. Immunolocalization experiments showed that SlY1 and SlX1 are localized in the nucleus, and that they are most abundant in cells that are actively dividing or beginning to differen-
Figure 1. Sexual dimorphism and Y chromosome in white campion (Silene latifolia). At a very early stage of development, the flower meristem contains all the organ primordia, as in hermaphrodite species : sepal (1), petal (2), stamen (3) and carpel (4). The subsequent development depends on the presence or absence of the Y chromosome. In an XY plant, female organ development is blocked by the female suppression function on the Y chromosome, and gives rise to a filament (f). The male organ (st) development is activated by the male promotion function on the Y chromosome. Finally, the male fertility genes located on the Y chromosome are involved in pollen development and maturation. In a XX plant, the female organ (gy) develops normally, as a default state, and male organs stop developing at a very early stage due to the lack of activation. PAR : pseudoautosomal region.
case for the human Y chromosome [12]? What is the extent of homology between the Silene latifolia X and Y chromosomes? Does the Y chromosome from Silene latifolia degenerate as expected for non-recombining chromosomes? What was the origin of the XY chromosomes in Silene?
2. Characterization of active genes on the Y chromosome In order to characterize active genes located on the Y chromosome from Silene latifolia, we microdissected Y
Figure 2. Expression of SlY1 and SlX1 at the cellular level. Longitudinal sections from different tissues were incubated with the purified antibodies recognizing SlY1 and SlX1 proteins. Bars, 100 µm for male flower and root tip, 500 µm for female flower and 50 µm for the ovules.
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F. Monéger / C.R. Acad. Sci. Paris, Sciences de la vie / Life Sciences 324 (2001) 531–535
Figure 3. Hypothesis concerning the evolution of sex chromosomes from a pair of autosomes. In a pair of autosomes, the crossing-over occurs during meiosis over all the length of the two chromosomes : it is represented by the dotted lines. The proto-XY evolve when there is cessation of the crossing-over in a portion of the autosomes pair containing at least two genes causing female sterility and male fertility (grey area filled with small black squares). During evolution, this portion where the two chromosomes do not pair during meiosis extends and finally, in the actual X and Y chromosomes, the pairing only occurs in the pseudo-autosomal region, small portion at the extremity of the chromosomes (filled with black).
tiate (figure 2). These results suggest a role of SLY1/SLX1 proteins in cell proliferation. Our sexual mutants deleted on the Y chromosome have lost the key genes involved in sex determination but still have SlY1. As a consequence, we can reasonably eliminate the possibility of a direct role of SlY1 in sex determination. The function of SlY1 being difficult to assess in Silene latifolia because of the lack of transformation system, the function of AtMSI4, a close homolog of SlY1 in Arabidopsis thaliana, is under investigation in our group. All our Y deletion mutants still contain the four other Y-linked genes suggesting that they are not directly involved in sex determination. However, it remains interesting to evaluate which kind of genes are located on the Y chromosome. For example, housekeeping genes as well as testis-specific genes have been found on the human Y chromosome [12]. What about the plant Y chromosome? For this reason, detailed analysis of the other Y-linked genes we have identified is in progress.
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3. Origin and evolution of plant sexual chromosomes Apart from the interest in sex determination, genes located on sex chromosomes provide an opportunity to investigate the molecular origin and evolution of sex chromosomes. The sex-linked genes we have characterized represent the tools required for such an analysis. The current hypothesis concerning the origin of sexual chromosomes is that they evolved from a pair of autosomes (figure 3). The initial events would be the appearance on a single chromosome of at least two genes causing female sterility and male fertility and the concomittant suppression of crossing-over at the corresponding loci. This cessation of recombination between the two chromosomes of the pair would slowly extend to reach almost all the chromosomes, as it is the case for the actual sex chromosomes (figure 3). A first analysis of SlY1 and SlX1 loci [18] showed that SlY1 is 20 times less polymorphic than SlX1,
F. Monéger / C.R. Acad. Sci. Paris, Sciences de la vie / Life Sciences 324 (2001) 531–535
confirming that different evolutionary constraints operate on the Y chromosome compared to the X. Comparison of the number of silent site substitutions between SlY1 and SlX1 allowed us to estimate that the two genes stopped recombining about 2.5·106 yr ago. However this is less than the putative age of the sex chromosomes. A possible explanation for this discrepancy is that the boundary between the pseudo-autosomal region and the nonrecombining sections of the Y chromosome has moved during the evolution of the sex chromosomes. Consequently, it is possible that different loci give different results. Currently, work is underway with the other sexlinked genes we have characterized in order to test this hypothesis. Another question we can assess is the origin of the sex chromosomes in the Silene family. For example, two closely related species of Silene latifolia also have sex chromosomes (Silene dioica and Silene diclinis) whereas another closely related species is hermaphrodite (Silene conica). It will be interesting to see if the sex-linked genes in S. latifolia are also sex-linked in S. dioica and S. diclinis.
References [1] Westergaard M., The relation between chromosome constitution and sex in the offspring of triploid Melandrium, Hereditas 34 (1948) 257–279. [2] Ainsworth C., Parker J., Buchanan-Wollaston V., Sex determination in plants, Curr. Top. Dev. Biol. 38 (1998) 167–223. [3] Parker J.S., Sex chromosomes and sexual differentiation in flowering plants, Chromosomes Today 10 (1990) 187–198. [4] Capel B., Sex in the 90s: SRY and the switch to the male pathway, Ann. Rev. Physiol. 60 (1998) 497–523. [5] Laurie C.C., The weaker sex is heterogametic: 75 years of Haldane’s rule, Genetics 147 (1997) 937–951. [6] Nöthiger R., Steinmann-Zwicky M., Results and problems in cell differentiation, structure and function of eukaryotic chromosomes, in: Hennig W. (Ed.), Genetics of sex determination in eucaryotes, Vol. 14, Springer-Verlag, Berlin, Heidelberg, 1987, pp. 271–300. [7] Raymond C.S., Shamu C.E., Shen M.M., Seifert K.J., Hirsch B., Hodgkin J., Zarkower D., Evidence for evolutionary conservation of sex-determining genes, Nature 391 (1998) 691–695. [8] Van Nigtevecht G., Genetic studies in dioecious Melandrium. II. Sex determination in Melandrium album and Melandrium dioicum, Genetica 37 (1966) 307–344. [9] Westergaard M., The mechanism of sex determination in dioecious flowering plants, Adv. Genet. 9 (1958) 217–281.
Additionally, the pair of automes in S. conica which carries these genes could be the ancestors of the sexchromosomes. This type of analysis is underway in our group. Analysis of the Y chromosome from Silene latifolia led us to investigate two main aspects : the first was the origin and evolution of sex chromosomes in plants. The second was the molecular mechanisms of early differentiation of reproductive organs, which seems to be conserved between phylogenetically distant species. As a consequence, we can predict that the study of sex determination in Silene latifolia will help to improve our understanding of early flower development in higher plants.
Acknowledgements. I would like to thank Professor Christian Dumas for supporting our work. I am grateful to Ioan Negrutiu for helpful discussions and to Mark Cock for correcting the manuscript. This work was supported by the CNRS, the INRA, the ENS Lyon and the University Lyon I.
[10] Farbos I., Oliveira M., Negrutiu I., Mouras A., Sex organ determination and differentiation in the dioecious plant Melandrium album (Silene latifolia): a cytological and histological analysis, Sex Plant Reprod. 10 (1997) 155–167. [11] Buzek J., Koutnikova H., Houben A., Riha K., Janousek B., Siroky J., Grant S., Vyskot B., Isolation and characterization of X chromosome-derived DNA sequences from a dioecious plant Melandrium album, Chromosome Res. 5 (1997) 57–65. [12] Lahn B.T., Page D.C., Functional coherence of the human Y chromosome, Science 278 (1997) 675–680. [13] Delichère C., Veuskens J., Hernould M., Barbacar N., Mouras A., Negrutiu I., Monéger F., SlY1, the first active gene cloned from a plant Y chromosome, encodes a WD-repeat protein, EMBO J. 18 (1999) 4169–4179. [14] Mulligan G., Jacks T., The retinoblastoma gene family: cousins with overlapping interests, Trends Genet. 14 (1998) 223–229. [15] Neer E.J., Schmidt C.J., Nambudripad R., Smith T.F., The ancient regulatory-protein family of WD-repeat proteins, Nature 371 (1994) 297–300. [16] Neer E.J., Smith T.F., G protein heterodimers: new structures propel new questions, Cell 84 (1996) 175–178. [17] Parkhurst S.M., Groucho: making its Marx as a transcriptional co-repressor, Trends Genet. 14 (1998) 130–132. [18] Filatov D.A., Monéger F., Negrutiu I., Charlesworth D., Low variability in a Y-linked plant gene and its implications for Y-chromosome evolution, Nature 404 (2000) 388–390.
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Review / Revue
Molecular and evolutionary analysis of a plant Y chromosome Françoise Monéger* Laboratoire de reproduction et développement des plantes, ENS Lyon, 46, allée d’Italie, 69364 Lyon cedex 07, France Received 23 October 2000; accepted 4 December 2000 Communicated by Christan Dumas
Abstract – Plants have evolved a great diversity of sex determination systems. Among these, the XY system, also found in mammals, is one of the most exciting since it gives the opportunity to compare the evolution of sex chromosomes in two different kingdoms. Whereas genetic and molecular mechanisms controlling sex determination in drosophila and mammals, have been well studied, very little is known about such processes in plants. White campion (Silene latifolia) is an example of plant with X and Y chromosomes. What is the origin of the X and Y chromosomes? How did they evolve from a pair of autosomes? In our laboratory, we have isolated the first active genes located on a plant Y chromosome. We are using them as markers to trace the origin and evolution of sex chromosomes in the Silene genus. © 2001 Académie des sciences/Éditions scientifiques et médicales Elsevier SAS sex chromosomes / Silene latifolia / molecular evolution / Y-linked genes / sex determination
Résumé – Analyse moléculaire et évolutive d’un chromosome Y végétal. Les plantes montrent une grande diversité dans leurs systèmes de détermination du sexe. Parmi eux, celui basé sur la présence de chromosomes XY semblables à ceux des mammifères est le plus attractif car il ouvre la possibilité de comparer l’évolution des chromosomes sexuels dans les deux règnes. Tandis que les mécanismes génétiques et moléculaires contrôlant la détermination du sexe ont été bien étudiés chez la drosophile et les mammifères, très peu de choses sont connues chez les plantes. Le compagnon blanc (Silene latifolia) est un exemple de plante modèle avec des chromosomes X et Y. Quelle est l’origine des chromosomes X et Y ? Comment ont-ils évolué à partir d’une paire d’autosomes ? Dans notre laboratoire, nous avons isolé les premiers gènes actifs localisés sur un chromosome Y végétal. Nous les utilisons aussi comme marqueurs pour retracer l’origine et l’évolution des chromosomes sexuels dans le genre Silene. © 2001 Académie des sciences/Éditions scientifiques et médicales Elsevier SAS chromosomes sexuels / Silene latifolia / évolution moléculaire / gènes liés au Y / détermination du sexe
. Version abrégée Les plantes montrent une grande diversité dans leurs systèmes de détermination du sexe. Parmi eux, celui basé sur la présence de chromosomes XY semblables à ceux des mammifères est le plus attractif car il ouvre la possibilité de comparer l’évolution des chromosomes
sexuels dans les deux règnes. Tandis que les mécanismes génétiques et moléculaires contrôlant la détermination du sexe chez la drosophile et les mammifères ont été bien étudiés, très peu de choses sont connues sur de tels processus chez les plantes. Le compagnon blanc (Silene latifolia) est un exemple de plante avec des chromosomes X et Y. À un stade très précoce, les
*Correspondence and reprints. E-mail address: [email protected] (F. Monéger).
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F. Monéger / C.R. Acad. Sci. Paris, Sciences de la vie / Life Sciences 324 (2001) 531–535
méristèmes floraux du compagnon blanc renferment à la fois les primordia d’organes mâles et femelles. Chez les plantes mâles, la présence d’un chromosome Y fonctionnel : 1) inhibe la prolifération cellulaire dans le territoire femelle, et 2) active le développement des organes mâles. Chez les plantes femelles (XX), le développement des organes femelles se déroule normalement (en l’absence d’inhibition), et l’absence d’activation mâle résulte dans l’arrêt du développement des organes mâles. Le chromosome Y porte aussi des gènes impliqués dans la fertilité mâle. Enfin, les chromosomes X et Y partagent une région pseudoautosomale (PAR) par laquelle ils s’apparient pendant la méiose. Quelle est l’origine des chromosomes X et Y ? Comment ont-ils évolué à partir d’une paire d’autosomes ? Dans notre laboratoire, nous avons isolé les premiers gènes actifs localisés sur un chromosome Y végétal. L’un d’eux (SlY1 pour ‘Silene latifolia gène
Y1’) code une protéine qui contient des domaines ‘WD-repeat’ et qui est vraisemblablement impliquée dans la prolifération cellulaire. Ce gène a un homologue sur le chromosome X (SlX1). Les deux gènes présentent un profil d’expression similaire, mis à part que SlY1 est absent des plantes femelles. La comparaison de séquences entre SlY1 et SlX1 nous permet d’estimer à quel moment les deux gènes ont cessé de recombiner, soit il y a environ 2,5 millions d’années. Ce genre d’analyse conduit sur plusieurs loci devrait nous permettre de dater l’émergence des chromosomes sexuels. Les cinq gènes que nous avons identifiés sur les chromosomes X et Y vont nous servir de marqueurs pour retracer l’histoire des chromosomes sexuels dans le genre Silene. Les chromosomes XY de plantes ont évolué beaucoup plus récemment que ceux des mammifères et nous donnent donc accès aux étapes initiales de l’évolution des chromosomes sexuels.
1. Introduction
cesses in plants. We have chosen to analyse sex determination in white campion (Silene latifolia) which is one of the most suitable plant species in which to address such questions. Sex determination in this Silene was reported, as in mammals, to be under the strict genetic control of a dominant Y chromosome [8, 9]. XX individuals develop female flowers, whereas XY individuals develop male flowers. The presence or absence of the Y chromosome determines the sex.
Most of the flowering plants are hermaphrodite. However, separation of sexes has been observed for a long time and was already mentioned in the 1889 book entitled The evolution of sex by P. Geddes and A. Thomson. At the beginning of the twentieth century, De Vries, Correns, Bateson, Baur, Shull and Winge made more or less extensive investigations in Silene latifolia : the presence of sex chromosomes in a plant , white campion (Silene latifolia, also called Melandrium album) was reported. In 1948, Westergaard demonstrated the active nature of the Y chromosome in white campion, by showing that a single Y chromosome is sufficient to induce the development of male flowers, even in the presence of several X chromosomes [1]. It is striking to note the ressemblance of this sex determination system to that of mammals. This convergent evolution of sex determination mechanisms raises exciting and challenging questions about structure, function and evolution of sex chromosomes in the two kingdoms. The majority of flowering plants (72 %) possess hermaphrodite flowers. However, different forms of sexuality exist : monoecy (separate unisexual flowers on the same plant, as in Zea mays) occurs in 7 % of higher plants; intermediates (both hermaphrodite and unisexual flowers on the same planta, as in Silene otites) which represent 17 %; and finally dioecy (unisexual flowers on separate plants, as in Asparagus officinalis) which occurs in 4 %. Some of the dioecious species have heteromorphic sex chromosomes. In animals, sex determination systems can be based on the X/autosome ratio (as in drosophila), or on X/Y with a dominant Y chromosome (as in mammals). Both systems exist in the plant kingdom [2, 3]. While genetic and molecular mechanisms controlling sex determination in drosophila and mammals have been extensively studied [4–7], very little is known about such pro-
532
At a very early stage, floral meristems from both sexes are potentially hermaphrodite : they contain both male and female reproductive organ primordia (figure 1). The first function on the Y chromosome to operate in male flowers is what we have called GSF (for gynoecium suppressing function). GSF is responsible for the sudden arrest of cell proliferation in the center of the flower meristem, which would otherwise develop into a gynoecium (female organ). The second function on the Y chromosome promotes stamen (male organ) development. As a result, in a male XY plant, flowers will develop with normal stamens and an undifferentiated filamentous structure instead of the gynoecium in the center of the flower (figure 1). Conversely, in a female XX plant, flowers will develop with a normal gynoecium but stamens will stop developing at an early stage [10]. The Y chromosome also carries male fertility genes (figure 1). The presence of these functions as well as their location on the Y chromosome, has been deduced from the sexual phenotype of mutants carrying Y chromosome deletions induced by gamma-irradiation. Finally, the X and Y chromosomes share a pseudoautosomal region (PAR) through which they specifically pair during meiosis [9, 11]. To date, our knowledge of the Y chromosome is limited to morphological, genetic and cytogenetic data. Several questions still remain to be answered. Does the Y chromosome from Silene latifolia, contain housekeeping genes as has been shown to be the
F. Monéger / C.R. Acad. Sci. Paris, Sciences de la vie / Life Sciences 324 (2001) 531–535
chromosomes on metaphase spreads from root cultures and used them in DOP-PCR (degenerated oligonucleotide primer–polymerase chain reaction) amplification. We used this Y-specific DNA to probe a cDNA library constructed from male flower buds. Among the positive clones, we isolated five cDNAs which hybridize to male-specific restriction fragments. These genes represent the first expressed genes identified on a plant Y chromosome. One of these genes, SlY1 (for Silene Latifolia Y1 gene), has been analysed in detail [13]. SlY1 is expressed predominantly in male flowers. A closely related gene, SlX1, is located on the X chromosome and is strongly expressed in both male and female flowers. SlY1 and SlX1 encode almost identical proteins containing WD-repeats. Such proteins are found almost exclusively in eukaryotes, where they are involved in a variety of processes including regulation of transcription, control of cell growth and differentiation, and chromatin structure [14–17]. Immunolocalization experiments showed that SlY1 and SlX1 are localized in the nucleus, and that they are most abundant in cells that are actively dividing or beginning to differen-
Figure 1. Sexual dimorphism and Y chromosome in white campion (Silene latifolia). At a very early stage of development, the flower meristem contains all the organ primordia, as in hermaphrodite species : sepal (1), petal (2), stamen (3) and carpel (4). The subsequent development depends on the presence or absence of the Y chromosome. In an XY plant, female organ development is blocked by the female suppression function on the Y chromosome, and gives rise to a filament (f). The male organ (st) development is activated by the male promotion function on the Y chromosome. Finally, the male fertility genes located on the Y chromosome are involved in pollen development and maturation. In a XX plant, the female organ (gy) develops normally, as a default state, and male organs stop developing at a very early stage due to the lack of activation. PAR : pseudoautosomal region.
case for the human Y chromosome [12]? What is the extent of homology between the Silene latifolia X and Y chromosomes? Does the Y chromosome from Silene latifolia degenerate as expected for non-recombining chromosomes? What was the origin of the XY chromosomes in Silene?
2. Characterization of active genes on the Y chromosome In order to characterize active genes located on the Y chromosome from Silene latifolia, we microdissected Y
Figure 2. Expression of SlY1 and SlX1 at the cellular level. Longitudinal sections from different tissues were incubated with the purified antibodies recognizing SlY1 and SlX1 proteins. Bars, 100 µm for male flower and root tip, 500 µm for female flower and 50 µm for the ovules.
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F. Monéger / C.R. Acad. Sci. Paris, Sciences de la vie / Life Sciences 324 (2001) 531–535
Figure 3. Hypothesis concerning the evolution of sex chromosomes from a pair of autosomes. In a pair of autosomes, the crossing-over occurs during meiosis over all the length of the two chromosomes : it is represented by the dotted lines. The proto-XY evolve when there is cessation of the crossing-over in a portion of the autosomes pair containing at least two genes causing female sterility and male fertility (grey area filled with small black squares). During evolution, this portion where the two chromosomes do not pair during meiosis extends and finally, in the actual X and Y chromosomes, the pairing only occurs in the pseudo-autosomal region, small portion at the extremity of the chromosomes (filled with black).
tiate (figure 2). These results suggest a role of SLY1/SLX1 proteins in cell proliferation. Our sexual mutants deleted on the Y chromosome have lost the key genes involved in sex determination but still have SlY1. As a consequence, we can reasonably eliminate the possibility of a direct role of SlY1 in sex determination. The function of SlY1 being difficult to assess in Silene latifolia because of the lack of transformation system, the function of AtMSI4, a close homolog of SlY1 in Arabidopsis thaliana, is under investigation in our group. All our Y deletion mutants still contain the four other Y-linked genes suggesting that they are not directly involved in sex determination. However, it remains interesting to evaluate which kind of genes are located on the Y chromosome. For example, housekeeping genes as well as testis-specific genes have been found on the human Y chromosome [12]. What about the plant Y chromosome? For this reason, detailed analysis of the other Y-linked genes we have identified is in progress.
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3. Origin and evolution of plant sexual chromosomes Apart from the interest in sex determination, genes located on sex chromosomes provide an opportunity to investigate the molecular origin and evolution of sex chromosomes. The sex-linked genes we have characterized represent the tools required for such an analysis. The current hypothesis concerning the origin of sexual chromosomes is that they evolved from a pair of autosomes (figure 3). The initial events would be the appearance on a single chromosome of at least two genes causing female sterility and male fertility and the concomittant suppression of crossing-over at the corresponding loci. This cessation of recombination between the two chromosomes of the pair would slowly extend to reach almost all the chromosomes, as it is the case for the actual sex chromosomes (figure 3). A first analysis of SlY1 and SlX1 loci [18] showed that SlY1 is 20 times less polymorphic than SlX1,
F. Monéger / C.R. Acad. Sci. Paris, Sciences de la vie / Life Sciences 324 (2001) 531–535
confirming that different evolutionary constraints operate on the Y chromosome compared to the X. Comparison of the number of silent site substitutions between SlY1 and SlX1 allowed us to estimate that the two genes stopped recombining about 2.5·106 yr ago. However this is less than the putative age of the sex chromosomes. A possible explanation for this discrepancy is that the boundary between the pseudo-autosomal region and the nonrecombining sections of the Y chromosome has moved during the evolution of the sex chromosomes. Consequently, it is possible that different loci give different results. Currently, work is underway with the other sexlinked genes we have characterized in order to test this hypothesis. Another question we can assess is the origin of the sex chromosomes in the Silene family. For example, two closely related species of Silene latifolia also have sex chromosomes (Silene dioica and Silene diclinis) whereas another closely related species is hermaphrodite (Silene conica). It will be interesting to see if the sex-linked genes in S. latifolia are also sex-linked in S. dioica and S. diclinis.
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Additionally, the pair of automes in S. conica which carries these genes could be the ancestors of the sexchromosomes. This type of analysis is underway in our group. Analysis of the Y chromosome from Silene latifolia led us to investigate two main aspects : the first was the origin and evolution of sex chromosomes in plants. The second was the molecular mechanisms of early differentiation of reproductive organs, which seems to be conserved between phylogenetically distant species. As a consequence, we can predict that the study of sex determination in Silene latifolia will help to improve our understanding of early flower development in higher plants.
Acknowledgements. I would like to thank Professor Christian Dumas for supporting our work. I am grateful to Ioan Negrutiu for helpful discussions and to Mark Cock for correcting the manuscript. This work was supported by the CNRS, the INRA, the ENS Lyon and the University Lyon I.
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