Immunohistochemical study of DNA methylation dynamics during plant development.

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.

Isolation and characterization of X chromosome-derived DNA sequences from a dioecious plant Melandrium album.

A number of X chromosome DNA sequences have been isolated from a dioecious plant, Melandrium album (syn. Silene latifolia), using chromosome microdissection followed by degenerate oligonucleotideprimed polymerase chain reaction (DOP-PCR) amplification. Six DNA clones were selected and further characterized by DNA/DNA hybridization techniques to check their copy numbers, sex-specific methylation patterns, species specificity and positions on chromosomes. These clones were moderately to highly repetitive (approximately 10(3)-10(5) copies per haploid genome) and none of them gave a positive signal on Northern blots. One of the clones yielded a sex-specific methylation pattern: its abundant non-methylated CCGG island was found only in males. All the clones also hybridized to two closely related dioecious Melandrium species (M. rubrum and M. dicline). Nucleotide sequences of two X-derived clones showed a number of internal short direct repeats; one of them strikingly resembled a plant conservative telomere sequence (TTTAGGG). None of the clones hybridized to the X chromosome only, but all were localized at the telomeric heterochromatic regions (DAPI C-bands) of both arms of a vast majority of M. album chromosomes using the fluorescence in situ hybridization (FISH) technique. However, the non-homologous arm of the Y chromosome (contrary to the arm homologous to the X chromosome, possessing the pseudoautosomal region) showed neither a DAPI C-banding-stained heterochromatin nor a FISH signal with any of the DNA probes tested, thus indicating its evolutionary diversification.

Epigenetic control of sexual phenotype in a dioecious plant, Melandrium album.

Melandrium album (syn. Silene latifolia) is a model dioecious species in which the Y chromosome, present only in heterogametic males, plays both a male-determining and a strict female-suppressing role. We showed that treatment with 5-azacytidine (5-azaC) induces a sex change to androhermaphroditism (an-dromonoecy) in about 21% of male plants, while no apparent phenotypic effect was observed in females. All of these bisexual androhermaphrodites (with the standard male 24, AA + XY karyotype) were mosaics possessing both male and hermaphrodite flowers and, moreover, the hermaphrodite flowers displayed various degrees of gynoecium development and seed setting. Southern hybridization analysis with a repetitive DNA probe showed that the 5-azacytidine-treated plants were significantly hypomethylated in CG doubles, but only to a minor degree in CNG triplets. The bisexual trait was transmitted to two successive generations, but only when androhermaphrodite plants were used as pollen donors. The sex reversal was inherited with incomplete penetrance and varying expressivity. Based on the uniparental inheritance pattern of androhermaphroditism we conclude that it originated either by 5-azaC induced inhibition of Y-linked female-suppressing genes or by a heritable activation of autosomal female-determining/promoting genes which can be reversed, on passage through female meiosis, by a genomic imprinting mechanism. The data presented indicate that female sex suppression in M. album XY males is dependent on methylation of specific DNA sequences and can be heritably modified by hypomethylating drugs.