Here we compare gene orders on the Silene latifolia sex chromosomes. On the basis of the deletion mapping results (11 markers and 23 independent Y chromosome deletion lines used), we conclude that a part of the Y chromosome (covering a region corresponding to at least 23.9 cM on the X chromosome) has been inverted. The gradient in silent-site divergence suggests that this inversion took place after the recombination arrest in this region. Because recombination arrest events followed by Y chromosome rearrangements also have been found in the human Y chromosome, this process seems to be a general evolutionary pathway.

The plant genus Silene has become a model for evolutionary studies of sex chromosomes and sex-determining mechanisms. A recent study performed in Silene colpophylla showed that dioecy and the sex chromosomes in this species evolved independently from those in Silene latifolia, the most widely studied dioecious Silene species. The results of this study show that the sex-determining system in Silene otites, a species related to S. colpophylla, is based on female heterogamety, a sex determination system that is unique among the Silene species studied to date. Our phylogenetic data support the placing of S. otites and S. colpophylla in the subsection Otites and the analysis of ancestral states suggests that the most recent common ancestor of S. otites and S. colpophylla was most probably dioecious. These observations imply that a switch from XX/XY sex determination to a ZZ/ZW system (or vice versa) occurred in the subsection Otites. This is the first report of two different types of heterogamety within one plant genus of this mostly nondioecious plant family. K E Y W O R D S :Evolution, sex chromosomes, sex determination, XY, ZW.

DNA methylation represents one of the key processes that play an important role in the transcriptional control of gene expression. The role of cytosine methylation in plant development has been demonstrated by at least three different kinds of evidence: parent-specific expression of some genes in developing seeds, control of flowering time and floral morphogenesis, and correlation with silencing of intrusive DNA sequences (mobile genetic elements and transgenes). In this work global changes in DNA methylation during seed germination and shoot apical meristem development in Silene latifolia have been studied using an indirect immunohistochemical approach. The data presented show that a rapid decrease in global DNA methylation during seed germination occurs first in endosperm tissue and subsequently in the hypocotyl. Using 5-bromo-2'-deoxyuridine pulses, it has been demonstrated that these demethylation events occurred before cell division had begun. In the early post-germination period, a decrease in DNA methylation was detected in cotyledons, also before cell division was observed. Taken together, these results indicate that DNA demethylation takes place in a non-replicative way, probably by an active mechanism. The central zone of the shoot apical meristem remains highly methylated during the whole period of vegetative growth and in this region, only a low cell division activity was found. However, upon the transition of the shoot apical meristem to the floral bud, the meristem both decreased its high methylation status and its cells started to divide. These data indicate that the central zone of the shoot apical meristem can represent a relatively quiescent 'germ-line' which is activated upon flowering to form spores and gametes.

The ends of eukaryotic chromosomes are capped with special nucleoprotein structures called telomeres. Telomere shortening due to telomerase inactivation may result in fusion of homologous or heterologous chromosomes, leading to their successive breakage during anaphase movement, followed by fusion of broken ends in the next cell cycle, i.e. the breakage-fusion-bridge (BFB) cycle. Using fluorescence in situ hybridization (FISH) with 25S rDNA and specific bacterial artificial chromosome (BAC) probes we demonstrate participation of chromosomes 2 and 4 of Arabidopsis thaliana AtTERT null plants in the formation of anaphase bridges. Both homologous and non-homologous chromosomes formed transient anaphase bridges whose breakage and unequal separation led to genome rearrangement, including non-reciprocal translocations and aneuploidy. The 45S rDNA regions located at the ends of chromosomes 2 and 4 were observed in chromosome bridges at a frequency approximately ten times higher than expected in the case of random fusion events. This outcome could result from a functional association of rDNA repeats at nucleoli. We also describe increased variation in the number of nucleoli in some interphase cells with supernumerary rDNA FISH signals. These data indicate that dysfunctional telomeres in Arabidopsis lead to massive genome instability, which is induced by multiple rounds of the BFB mechanism.

Understanding the origin and evolution of sex chromosomes requires studying recently evolved X-Y chromosome systems such as those in some flowering plants. We describe Y chromosome deletion mutants of Silene latifolia, a dioecious plant with heteromorphic sex chromosomes. The combination of results from new and previously described deletions with histological descriptions of their stamen development defects indicates the presence of two distinct Y regions containing loci with indispensable roles in male reproduction. We determined their positions relative to the two main sex determination functions (female suppressing and the other male promoting). A region proximal to the centromere on the Y p arm containing the putative stamen promoting sex determination locus includes additional early stamen developmental factors. A medial region of the Y q arm carries late pollen fertility factors. Cytological analysis of meiotic X-Y pairing in one of the male-sterile mutants indicates that the Y carries sequences or functions specifically affecting sex chromosome pairing.

Switches in heterogamety are known to occur in both animals and plants. Although plant sex determination systems probably often evolved more recently than those in several well-studied animals, including mammals, and have had less time for switches to occur, we previously detected a switch in heterogamety in the plant genus Silene: section Otites has both female and male heterogamety, whereas S. latifolia and its close relatives, in a different section of the genus, Melandrium (subgenus Behenantha), all have male heterogamety. Here we analyse the evolution of sex chromosomes in section Otites, which is estimated to have evolved only about 0.55 MYA. Our study confirms female heterogamety in S. otites and newly reveals female heterogamety in S. borysthenica. Sequence analyses and genetic mapping show that the sex-linked regions of these two species are the same, but the region in S. colpophylla, a close relative with male heterogamety, is different. The sex chromosome pairs of S. colpophylla and S. otites each correspond to an autosome of the other species, and both differ from the XY pair in S. latifolia. Silene section Otites species are suitable for detailed studies of the events involved in such changes, and our phylogenetic analysis suggests a possible change from female to male heterogamety within this section. Our analyses suggest a possibility that has so far not been considered, change in heterogamety through hybridization, in which a male-determining chromosome from one species is introgressed into another one, and over-rides its previous sex-determining system.

BackgroundPrior to this study, no differences in gene expression between male and female dioecious plants in the vegetative state had been detected. Among dioecious plants displaying sexual dimorphism, Silene latifolia is one of the most studied species. Although many sexually dimorphic traits have been described in S. latifolia, all of them are quantitative, and they usually become apparent only after the initiation of flowering.ResultsWe present RT-PCR-based evidence that in S. latifolia, sexual dimorphism in gene expression is present long before the initiation of flowering. We describe three ESTs that show sex-specific (two male specific and one female specific) transcription at the rosette stage before the first flowering season.ConclusionsTo our knowledge, this study provides the first molecular evidence of early pre-flowering sexual dimorphism in angiosperms.

Summary.The first pollen mitosis results in generative and vegetative cells which are characterised by a striking difference in their chromatin structure. In this study, histone H4 acetylation and DNA methylation have been analysed during pollen development in Lilium longiflorum. Indirect immunofiuorescence procedures followed by epiftuorescence and taser scanning microscopy enabled a relative quantification of H4 acetylation and DNA methylation in microspores, immature binucleate pollen, mature pollen, and pollen tubes. The results show that histone H4 of the vegetative nucleus, in spite of its decondensed chromatin structure, is strongly hypoacetylated at lysine positions 5 and 8 in comparison with both the original microspore nucleus and the generative-cell nucleus. These H4 terminal lysines in the vegetative nucleus are, however, progressively acetylated during the following pollen tube growth. The DNA methylation analysis inversely correlates with the histone acetylation data. The vegetative nucleus in mature pollen grains is heavily methylated, but a dramatic nonreplicative demethylation occurs during the pollen tube development. Changes neither in H4 acetylation nor in DNA methylation have been found during development of the generative nucleus. The results obtained indicate that the vegetative nucleus enters the quiescent state (accompanied by DNA hypermethylation and H4 underacetylation) during the maturation of pollen grain which enables pollen grains a long-term survival without external source of nutrients until they reach the stigma.