Dioecious Plant Silene Latifolia Research Articles

Ultrastructural analysis of the behavior of the dimorphic fungus Microbotryum violaceum in fungus-induced anthers of female Silene latifolia flowers

The development of male organs is induced in female flowers of the dioecious plant Silene latifolia by infection with the fungus Microbotryum violaceum. Stamens in a healthy female flower grow only to stage 6, whereas those in an infected female flower develop to the mature stage (stage 12), at which the stamens are filled with fungal teliospores instead of pollen grains. To investigate these host-parasite interactions, young floral buds and fungus-induced anthers of infected female flowers were examined by electron microscopy following fixation by a high-pressure freezing method. Using this approach, we found that parasitic hyphae of this fungus contain several extracellular vesicles and have a consistent appearance up to stage 8. At that stage, parasitic hyphae are observed adjacent to dying sporogenous cells in the infected female anther. At stage 9, an increased number of dead and dying sporogenous cells is observed, among which the sporogenous hyphae of the fungus develop and form initial teliospores. Several types of electron-dense material are present in proximity to some fungi at this stage. The initial teliospores contain two types of vacuoles, and the fungus cell wall contains abundant carbohydrate, as revealed by silver protein staining. The sporogenous cell is probably sensitive to infection by the fungus, resulting in disruption. In addition, the fungus accelerates cell death in the anther and utilizes constituents of the dead host cell to form the mature teliospore.

An anther- and petal-specific gene SlMF1 is a multicopy gene with homologous sequences on sex chromosomes

A male flower-specific gene SlMF1 was isolated from male flower buds of the dioecious plant Silene latifolia. SlMF1 is expressed in all the floral meristems at the very early stage of development in both male and female flower buds. At the mature stage of development in male flower buds, SlMF1 transcripts were specifically accumulated in pollen mother cells, tapetal cells, and the developing tips of petals. Genomic Southern hybridization revealed that SlMF1 was a multicopy gene with a Y chromosome-linked homologous sequence. PCR analyses with flow-sorted chromosomes showed that SlMF1 was localized on both autosomes and the X chromosome.

Characterization of two SEPALLATA MADS-box genes from the dioecious plant Silene latifolia

Two SEPALLATA orthologs, SlSEP1 and SlSEP3, were isolated from male flower buds of the dioecious plant Silene latifolia. Both genes are located on autosomes and not on the sex chromosomes. SlSEP1 and SlSEP3 transcripts were detected specifically in male and female flower buds. Quantitative RT-PCR and in situ hybridization revealed that both genes were expressed in young flower meristems, developing petals, male anthers, and female ovules. However, neither transcript was detected in suppressed gynoecia of male flower buds or suppressed anthers of female flower buds. The genes were differentially expressed during anther development, with the SlSEP1 transcript accumulating in anther walls and tapetal cells, and the SlSEP3 transcript accumulating in tetrads as well as anther walls and tapeta. Transcripts of both genes were also detected in flower buds of mutants with deletions of some parts of the Y chromosome, suggesting that neither gene is directly involved in sex determination.

GENETIC CONSTRAINTS ON FLORAL EVOLUTION IN A SEXUALLY DIMORPHIC PLANT REVEALED BY ARTIFICIAL SELECTION

Sexual dimorphism is one of the most widespread and recognizable patterns of phenotypic variation in the biotic world. Sexual dimorphism in floral display is striking in the dioecious plant Silene latifolia, with males making many, small flowers compared to females. We investigated this dimorphism via artificial selection on two populations to determine whether genetic variation exists within populations for flower size and the extent of the between-sex correlation, whether a flower size and number trade-off exists within each sex, and whether pollen and ovule production vary with flower size. We selected for decreased flower size (calyx width) in females and increased flower size in males and measured the response to selection in size and correlated responses in flower dry mass, flower number, and pollen or ovule number per flower. Four bouts of selection in each of two selection programs were performed, for a total of three selection lines to decrease size, three to increase it, and two control lines. Flower size always significantly responded to selection and we always found a significant correlated response in the sex not under selection. Selection decreased but did not eliminate the sexual dimorphism in flower dry mass and number. A negative relationship between flower size and number within each sex was revealed. Whereas ovule number showed a significant correlated response to selection on flower size, pollen number did not. Our results indicate that although substantial additive genetic variation for flower size exists, the high between-sex genetic correlation would likely constrain flower size from becoming more sexually dimorphic. Furthermore, floral display within each sex is constrained by a flower size and number trade-off. Given this trade-off and lack of variation in pollen production with flower size, we suggest that sexual dimorphism evolved via sexual selection to increase flower number in males but not females.

Sex-specific cell division during development of unisexual flowers in the dioecious plant Silene latifolia.

We analyzed cell division patterns during the differentiation of unisexual flowers of the dioecious plant Silene latifolia using in situ hybridization with histone H4 and cyclin A1 genes. The gene expression patterns indicated that the activation of cell divisions in whorls 3 and 4 was reversed in young male and female flower buds. During maturation of flower buds, a remarkable reduction in cell division activity occurred in the male gynoecium primordium and female stamen primordia. Our analyses showed that differential activation and reduction of cell division strongly correlated with sex-specific promotion and cessation in the sex differentiation of unisexual flowers.

GENETIC CONSTRAINTS ON FLORAL EVOLUTION IN A SEXUALLY DIMORPHIC PLANT REVEALED BY ARTIFICIAL SELECTION

Sexual dimorphism is one of the most widespread and recognizable patterns of phenotypic variation in the biotic world. Sexual dimorphism in floral display is striking in the dioecious plant Silene latifolia, with males making many, small flowers compared to females. We investigated this dimorphism via artificial selection on two populations to determine whether genetic variation exists within populations for flower size and the extent of the between-sex correlation, whether a flower size and number trade-off exists within each sex, and whether pollen and ovule production vary with flower size. We selected for decreased flower size (calyx width) in females and increased flower size in males and measured the response to selection in size and correlated responses in flower dry mass, flower number, and pollen or ovule number per flower. Four bouts of selection in each of two selection programs were performed, for a total of three selection lines to decrease size, three to increase it, and two control lines. Flower size always significantly responded to selection and we always found a significant correlated response in the sex not under selection. Selection decreased but did not eliminate the sexual dimorphism in flower dry mass and number. A negative relationship between flower size and number within each sex was revealed. Whereas ovule number showed a significant correlated response to selection on flower size, pollen number did not. Our results indicate that although substantial additive genetic variation for flower size exists, the high between-sex genetic correlation would likely constrain flower size from becoming more sexually dimorphic. Furthermore, floral display within each sex is constrained by a flower size and number trade-off. Given this trade-off and lack of variation in pollen production with flower size, we suggest that sexual dimorphism evolved via sexual selection to increase flower number in males but not females.

Morphological development of anthers induced by the dimorphic smut fungus Microbotryum violaceum in female flowers of the dioecious plant Silene latifolia.

When inoculated with the dimorphic smut fungus Microbotryum violaceum (Pers.) G. Deml and Oberwinkler, the female flower of the dioecious plant Silene latifolia (Miller) E.H.L. Krause develops anther-like structures filled with spores instead of pollen grains. Using natural scanning electron microscopy, Nomarski interference microscopy, and fluorescence microscopy, we investigated the morphological modifications of the host plant resulting from this parasitism and the localization of smut hyphae in the flower bud. Flowers of infected plants lasted significantly longer than those of healthy plants, probably because the infection strengthened floral organs, such as the flower base and the anther filaments. Smut hyphae were observed throughout all organs of the young flower buds of infected plants, including sepals, petals, stamens, and pistil primordia. In healthy female flowers, anthers initiated sporogenous cell formation, but lacked parietal cell layers. By contrast, the parietal cell layers of infected female flowers differentiated into tapetal tissue, middle cell layers, and endothecial layers, as in the anthers of healthy male flowers. Smut spore formation in the infected anther was initiated in intercellular regions between the sporogenous cells, resulting in degeneration of premature sporogenous cells, tapetal tissue, and middle cell layers. The development of the endothecial layers and epidermis in the infected anther were morphologically normal.

The Sex Chromosomes of Silene latifolia Revisited and Revised

Classical studies have established that, during meiosis, the X and Y chromosomes of the model dioecious plant Silene latifolia pair over a region at the ends of their q arms. We used fluorescence in situ hybridization of two molecular markers to demonstrate that this widely accepted model is incorrect. From these data we conclude that the homologous arm of the X chromosome is the p arm and that of the Y chromosome is the q arm. The establishment of the proper orientation of the pseudoautosomal region is essential for mapping and evolutionary studies.

Organization of the KpnI family of chromosomal distal-end satellite DNAs in Silene latifolia.

The major satellite DNAs of the dioecious plant Silene latifolia are represented by the repetitive sequences X43.1, RMY1 and members of the SacI family, which are located at the distal ends of chromosomes. To characterize the satellite DNAs at the distal ends of the chromosomes in S. latifolia ( Sl-distal-satDNA), we isolated a bacterial artificial chromosome clone (number 15B12) that contained multiple repeat sequences with KpnI restriction sites, and subcloned a portion of this sequence into a plasmid vector. Sequencing analysis confirmed that recognition or degenerate sites for KpnI were repeated 26 times at intervals of 310-324 bp in the inserted DNA. The phylogenetic tree that was constructed with the 26 KpnI repeat units contained clustered branches that were independent of the SacI family. It is clear that the KpnI repeat belongs to an Sl-distal-satDNA family that is distinct from the SacI family. We designated this family as " KpnI" after the restriction enzyme that does not have a site in the SacI family. Multi-colored fluorescent in situ hybridization was performed with the KpnI family and RMY1 probes under high stringency conditions. The results suggest that chromosome 7 is unique and that it carries the KpnI family at only one end.

Genetic and functional analysis of DD44, a sex-linked gene from the dioecious plant Silene latifolia, provides clues to early events in sex chromosome evolution.

Genetic and functional analysis of DD44, a sex-linked gene from the dioecious plant Silene latifolia, provides clues to early events in sex chromosome evolution.

Isolation and characterization of two homeodomain leucine zipper genes from the dioecious plant Silene latifolia.

Homeodomain leucine zipper (HD-Zip) genes encode transcription factors that are characterized by both a homeodomain and a leucine zipper motif. Two HD-Zip genes were isolated from cDNA of the male flower bud of the dioecious plant Silene latifolia. The two isolated genes, SlHDL1 and SlHDL2, encode proteins with the characteristics of HD-Zip transcription factors belonging to HD-Zip classes I and II, respectively. The expression patterns of SlHDL1 and SlHDL2 throughout the floral developmental stages were studied using real-time PCR and in situ hybridization. SlHDL1 is specifically expressed in the outermost layer of the anthers and gynoeciums with a patchy pattern in the inner layers, suggesting that the product of SlHDL1 plays a role in the early developmental stage of the epidermal tissues of these floral organs. Its expression pattern in the anthers and gynoeciums suggests an involvement in differentiation of the reproductive organs. On the other hand, real-time PCR revealed accumulation of SlHDL2 transcripts in the anther and pollen grains of the male flower. These results suggest that SlHDL1 and SlHDL2 regulate specific targets in restricted regions leading to floral organ differentiation in S. latifolia.

CCLS96.1, a member of a multicopy gene family, may encode a non-coding RNA preferentially transcribed in reproductive organs of Silene latifolia.

Dioecy in the model dioecious plant Silene latifolia is determined genetically by its heteromorphic sex chromosomes. A bacterial artificial chromosome (BAC) clone, #19B12, was isolated by screening a BAC library from S. latifolia using polymerase chain reaction (PCR) with a set of sequence tagged site (STS) primers, ScD05, which are specific to the Y chromosome. A portion of #19B12 was subcloned to construct plasmid #25-1, with an insert of 7.8 kb. This 7.8-kb fragment encodes ScD05 homolog and an anther-specific gene, CCLS96.1. Northern blot analysis of CCLS96.1 indicated a faint band of 1.8 kb in male and female flower buds. 5' and 3' rapid amplification of cDNA ends (RACE) indicated that transcripts of CCLS96.1 are very varied in size. Moreover, semi-quantitative reverse transcription-PCR (RT-PCR) showed that CCLS96.1 was also expressed in both male and female leaves. RACE produced at least ten species of transcripts, with 79-97% similarity among them. However, no significant ORFs could be predicted from their nucleotide sequences, since each has numerous stop codons throughout all three reading frames. Genomic Southern hybridization showed that the S. latifolia genome contains numerous CCLS96.1 homologs. These results suggest that the transcripts of CCLS96.1 play some role as multiple non-coding RNAs in S. latifolia.

Among-population variation and correlations in sexually dimorphic traits of Silene latifolia

Abstract The degree of sexual dimorphism in a trait may be determined directly by disruptive selection, as well as by correlations with other traits under selection. We grew seeds from nine populations of the dioecious plant Silene latifolia in a common-garden experiment to determine whether phenotypic variation and correlations existed for floral, leaf and resource allocation traits, and whether this variation had a genetic component. We also determined the traits which were sexually dimorphic, the degree of dimorphism, and whether it varied among populations. Seven traits exhibited among-population variation and sexual dimorphism. Variation in the degree of dimorphism occurred only for two traits, suggesting that dimorphism may be evolving more slowly than trait means. Males had more, smaller flowers, shorter leaves, and allocated less of their total biomass to stems and more to leaves than females. Flower production was the most sexually dimorphic trait and was correlated with all measured traits. Most traits exhibited significant correlations between the sexes. The pattern of correlations and the degree of sexual dimorphism among traits lead us to suggest that intrasexual selection for an exaggerated floral display in males has indirectly led to sexual dimorphism in a host of other traits.

Distribution of interstitial telomere-like repeats and their adjacent sequences in a dioecious plant, Silene latifolia.

The dioecious plant Silene latifolia has large, heteromorphic X and Y sex chromosomes that are thought to be derived from rearrangements of autosomes. To reveal the origin of the sex chromosomes in S. latifolia, we isolated and characterized telomere-homologous sequences from intra-chromosomal regions (interstitial telomere-like repeats; ITRs) and ITR-adjacent sequences (IASs). Nine genomic DNA fragments with degenerate 84- to 175-bp ITRs were isolated from a genomic library and total genome of male plants. Comparing the nucleotide sequences, the IASs of the nine ITRs were classified into seven elements (IAS-a, IAS-b, IAS-c, IAS-d, IAS-e, IAS-f, and IAS-g) by sequence similarity. The ITRs were grouped into two classes (class-I and -II ITRs) according to the classification of IASs. The class-I ITRs were sub-grouped into three subclasses (subclasses-IA, -IB, and -IC ITRs) based on the arrangement of IAS elements. By contrast, the class-II ITR was located between two different IASs (IAS-f and IAS-g). Genomic Southern analyses showed that both the male and female genomes contained six (IAS-f) to 153 (IAS-d) copies of each IAS per haploid genome. Fluorescence in situ hybridization analyses showed that one IAS element, IAS-d, was distributed in the interstitial and proximal regions of the sex chromosomes of S. latifolia. The distribution of IAS-d is important evidence for past telomere-mediated chromosome rearrangements during the evolution of the sex chromosomes of S. latifolia.

LTR retrotransposons in the dioecious plant Silene latifolia.

Conserved domains of two types of LTR retrotransposons, Tyl-copia- and Ty3-gypsy-like retrotransposons, were isolated from the dioecious plant Silene latifolia, whose sex is determined by X and Y chromosomes. Southern hybridization analyses using these retrotransposons as probes resulted in identical patterns from male and female genomes. Fluorescence in situ hybridization indicated that these retrotransposons do not accumulate specifically in the sex chromosomes. These results suggest that recombination between the sex chromosomes of S. latifolia has not been severely reduced. Conserved reverse transcriptase regions of Ty1-copia-like retrotransposons were isolated from 13 different Silene species and classified into two major families. Their categorization suggests that parallel divergence of the Ty1-copia-like retrotransposons occurred during the differentiation of Silene species. Most functional retrotransposons from three dioecious species, S. latifolia, S. dioica, and S. diclinis, fell into two clusters. The evolutionary dynamics of retrotransposons implies that, in the genus Silene, dioecious species evolved recently from gynodioecious species.

Male specific genes from dioecious white campion identified by fluorescent differential display.

Fluorescent differential display (FDD) has been used to screen for cDNAs that are differentially up-regulated in male flowers of the dioecious plant Silene latifolia in which an X/Y chromosome system of sex determination operates. To adapt FDD to the cloning of large numbers of differential cDNAs, a novel method of confirming the differential expression of these has been devised. FDD gels were Southern electro-blotted and probed with mixtures of individual cDNA clones derived from different FDD product ligation reactions. These Southern blots were then stripped and re-probed with further mixtures of individual cloned FDD products to identify the maximum number of recombinant clones carrying the true differential amplification products. Of 135 differential bands identified by FDD, 56 differential amplification products were confirmed; these represent 23 unique differentially expressed genes as determined by virtual Northern analysis and two genes expressed at or below the level of detection by virtual Northern analysis. These two low expressed genes show bands of hybridization on genomic Southern blots that are specific to male plants, indicating that they are derived from, or closely related to, Y chromosome genes.

A plant Y chromosome-STS marker encoding a degenerate retrotransposon.

The dioecious plant Silene latifolia has both X and Y sex chromosomes. Male-specific random amplified polymorphic DNA (RAPD) fragments were analyzed to identify Y-chromosome-linked sequences. One of the RAPD fragments, MS4, was converted into a more reliable and reproducible sequence-tagged site (STS) marker. A set of MS4 STS primers was used to amplify two genomic DNA fragments (MS4a and MS4b) from a male plant and one (MS4a) from a female plant, which indicates that MS4b is located on the Y chromosome. Sequence analysis revealed that MS4a encoded a gag protein of a Ty3-gypsy-like retrotransposon. A 147-bp region from the middle of MS4a was deleted in MS4b. The MS4b sequence was not detected in the most closely related dioecious species, S. dioica. This suggests that a retrotransposon with the MS4b sequence has degenerated recently on the Y chromosome.

Euchromatic domains in plant chromosomes as revealed by H4 histone acetylation and early DNA replication

Using specific polyclonal antisera raised against acetylated isoforms of histone H4, we have analyzed their distribution in the dioecious plant Silene latifolia (syn. Melandrium album) possessing heteromorphic sex chromosomes. Our previous studies on this species have shown that one of the two X chromosomes in homogametic female cells is heavily methylated and late replicating, as a possible consequence of dosage compensation. Here we report that there are no detectable differences in intensity and distribution of H4 acetylation between these two X chromosomes. In S. latifolia only distal-subtelomeric chromosome regions, on both the sex chromosomes and autosomes, display strong signals of H4 acetylation at N-terminal lysines 5, 8, and 12. These acetylated domains correspond to the very early replicating distal chromosome regions as revealed by 5-bromodeoxyuridine pulses followed by the indirect immunofluorescence microscopy. The distribution of H4 acetylated at lysine 16 was uniform along the chromosomes. The unique distal-subtelomeric H4 acetylation signals were also observed in three other Silene species (S. vulgaris, S. pendula, and S. chalcedonica), but not in two non-related plant species tested (Allium cepa and Nicotiana tobacum). The presented data as well as our recent studies on the structure of S. latifolia chromosome ends indicate that Silene species possess the specific distal-subtelomeric location of euchromatin, gene-rich regions on chromosomes.

An X-linked gene with a degenerate Y-linked homologue in a dioecious plant.

Most flowering plants are hermaphroditic, having flowers with both male and female parts. Less than 4% of plant species are dioecious (with individuals of separate sexes), and many of these species have chromosome-mediated sex determination. The taxonomic distribution of separate sexes and chromosomal sex-determination systems in the flowering plants indicates that plant sex chromosomes have evolved recently through replicated, independent events, contrasting with the ancient origins of mammalian and insect sex chromosomes. Plant sex chromosomes, therefore, offer opportunities to study the most interesting early stages of the evolution of sex chromosomes. Here we show that a gene encoding a male-specific protein is linked to the X chromosome in the dioecious plant Silene latifolia, and that it has a degenerate homologue in the non-pairing region of the Y chromosome. The Y-linked locus has degenerated as a result of nucleotide deletion and the accumulation of repetitive sequences. We have identified both the first X-linked gene and the first pair of homologous sex-linked loci to be found in plants. The homology between the active X-linked locus and the degenerate Y-linked locus supports a common ancestry for these two loci.

Male reproductive organ-specific genes in the dioecious plant Silene latifolia

Male reproductive organ-specific genes in the dioecious plant Silene latifolia