The content and distribution of the poly(A) RNA and splicing machinery element--TMG snRNA in cells of the Hyacinthus orientalis L. mature embryo sac, during the progamic phase and after fertilization, were investigated. Using fluorescence in situ hybridization and immunofluorescence methods we showed that in the mature unfertilized embryo sac strong signal indicating poly(A) RNA and TMG snRNA appeared only in synergids and antipodal cells. In synergids accumulation of poly(A) RNA was shown in cytoplasm around the filiform apparatus. In the egg cell and central cell of the embryo sac accumulation of poly(A) RNA was very low and only low pool of TMG snRNA was observed in the nuclei of these cells. During the progamic phase, dramatic changes in the accumulation and distribution of poly(A) RNA and TMG snRNA were observed in the synergids. In these cells a considerable decrease in accumulation of TMG snRNA in the nucleus and poly(A) RNA in the region of the filiform apparatus occurred. In the egg cell and central cell the content of polyadenylated transcripts and TMG snRNA was still low. After fertilization a drastic increase in polyadenylated transcripts and TMG snRNA content was observed in the cells which undergo a fusion with the sperm cells (the zygote and the fertilized central cells). In contrast, a progressive decrease in poly(A) RNA and splicing snRNAs accumulation was observed in degenerating antipodal cells and synergids.

We characterized three phases of Hyacinthus orientalis L. embryo sac development, in which the transcriptional activity of the cells differed using immunolocalization of incorporated 5′-bromouracil, the total RNA polymerase II pool and the hypo- (initiation) and hyperphosphorylated (elongation) forms of RNA Pol II. The first stage, which lasts from the multinuclear stage to cellularization, is a period of high transcriptional activity, probably related to the maturation of female gametophyte cells. The second stage, encompassing the period of embryo sac maturity and the progamic phase, involves the transcriptional silencing of cells that will soon undergo fusion with male gametes. During this period in the hyacinth egg cell, there are almost no newly formed transcripts, and only a small pool of RNA Pol II is present in the nucleus. The transcriptional activity of the central cell is only slightly higher than that observed in the egg cell. The post-fertilization stage is related to the transcriptional activation of the zygote and the primary endosperm cell. The rapid increase in the pool of newly formed transcripts in these cells is accompanied by an increase in the pool of RNA Pol II, and the pattern of enzyme distribution in the zygote nucleus is similar to that observed in the somatic cells of the ovule. Our data, together with the earlier results of Pięciński et al. (2008), indicate post-fertilization synthesis and the maturation of numerous mRNA transcripts, suggesting that fertilization in H. orientalis induces the activation of the zygote and endosperm genomes.

The nucleolar activity of Hyacinthus orientalis L. embryo sac cells was investigated. The distributions of nascent pre-rRNA (ITS1), 26S rRNA and of the 5S rRNA and U3 snoRNA were determined using fluorescence in situ hybridization (FISH). Our results indicated the different rRNA metabolism of the H. orientalis female gametophyte cells before and after fertilization. In the target cells for the male gamete, i.e., the egg cell and the central cell whose activity is silenced in the mature embryo sac (Pięciński et al. in Sex Plant Reprod 21:247–257, 2008; Niedojadło et al. in Planta doi:10.1007/s00425-012-1599-9, 2011), rRNA metabolism is directed at the accumulation of rRNPs in the cytoplasm and immature transcripts in the nucleolus. In both cells, fertilization initiates the maturation of the maternal pre-rRNA and the expression of zygotic rDNA. The resumption of rRNA transcription observed in the hyacinth zygote indicates that in plants, there is a different mechanism for the regulation of RNA Pol I activity than in animals. In synergids and antipodal cells, which have somatic functions, the nucleolar activity is correlated with the metabolic activity of these cells and changes in successive stages of embryo sac development.Electronic supplementary materialThe online version of this article (doi:10.1007/s00425-012-1618-x) contains supplementary material, which is available to authorized users.