Homogalacturonan deesterification during pollen–ovule interaction in Larix decidua Mill.: an immunocytochemical study

Studies on angiosperm plants have shown that homogalacturonan present in the extracellular matrix of pistils plays an important role in the interaction with the male gametophyte. However, in gymnosperms, knowledge on the participation of HG in the pollen–ovule interaction is limited, and only a few studies on male gametophytes have been reported. Thus, the aim of this study was to determine the distribution of HG in male gametophytes and ovules during their interaction in Larix decidua Mill. The distribution of HG in pollen grains and unpollinated and pollinated ovules was investigated by immunofluorescence techniques using monoclonal antibodies that recognise high methyl-esterified HG (JIM7), low methyl-esterified HG (JIM5) and calcium cross-linked HG (2F4). All studied categories of HG were detected in the ovule. Highly methyl-esterified HG was present in the cell walls of all cells throughout the interaction; however, the distribution of low methyl-esterified and calcium cross-linked HG changed during the course of interaction. Both of these categories of HG appeared only in the apoplast and the extracellular matrix of the ovule tissues, which interact with the male gametophyte. This finding suggests that in L. decidua, low methyl-esterified and calcium cross-linked HG play an important role in pollen–ovule interaction. The last category of HG is most likely involved in adhesion between the pollen and the ovule and might provide an optimal calcium environment for pollen grain germination and pollen tube growth

Significance of selenium supplementation in root‑ shoot reactions under manganese stress in wheat seedlings– biochemical and cytological studies

Purpose Agronomic practices are one of the reasons for the increasing accumulation of elements in the soil, including manganese (Mn). Our previous studies have shown that selenium (Se) ions can reduce the toxic actions of metal stress. Those, we studied the effects of Mn—treated as a stressor and Se – as a potential defense in plants. Methods Mn ions (10 mM) or/and Se (15 μM) were added into hydroponic nutrients of two wheat cultivares. The evaluation of the stress-generating and protective actions were analyzed by biochemical methods and microscopic observations in leaves and roots. Moreover the level of DNA methylation for these tissues was determined. Results Mn application caused an increase of lipid peroxidation and hydrogen peroxide content in both leaves and roots and was accompanied with a greater absorption of this element by the roots. For other elements (K, Fe, S, P), with the exception of Ca, the reduced their uptake was registered, especially in roots. For roots, Mn stimulated greater, microscopically observed, desorganization in cell structure as compared to leaves, which was accompanied by a quantitative increase in 5-methylcytosine (5-metC) in root meristem. Se application diminished the effects of Mn-stress. Conclusions These studies is the first in which indicated that global 5-metC level in roots enhancing from dividing meristematic cells to elongating cells of the axial cylinder and cortex. It was suggested that the rise in Ca level can lead to modification of root cells differentiations what may be one of the steps in defense mechanisms

Ribosomal RNA of Hyacinthus orientalis L. female gametophyte cells before and after fertilization

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

Transcriptional activity of Hyacinthus orientalis L. female gametophyte cells before and after fertilization

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

Distribution of exchangeable Ca2+ during the process of Larix decidua Mill. pollination and germination

Abstract The involvement of Ca2+ ions in angiosperms sexual processes is well established, while in gymnosperms, such knowledge remains limited and is still a topic of discussion. In this study, we focused on Larix decidua, using Alizarin-red S staining and the pyroantimonate method to examine the tissue and subcellular distribution of free and loosely bound Ca2+ ions at different stages of the male gametophyte's development and its interaction with the ovule. Our findings show that in larch, both the germination of pollen grains and the growth of pollen tubes occur in an environment rich in Ca2+. These ions play a crucial role in the adhesion of the pollen grain to the stigmatic tip and its subsequent movement to the micropylar canal. There is a significant presence of free and loosely bound Ca2+ ions in both the fluid of the micropylar canal and the extracellular matrix of the nucellus. As the pollen tube extends through the nucellus, we observed a notable accumulation of Ca2+ ions just above the entry to the mature archegonium, a region likely crucial for the male gametophyte's directional growth. Meanwhile, the localized presence of free and loosely bound Ca2+ ions within the egg cell cytoplasm may inhibit the pollen tubes growth and rupture, playing an important role in fertilization

Spatial and Temporal Distribution of Arabinogalactan Proteins during Larix decidua Mill. Male Gametophyte and Ovule Interaction

The role of ArabinoGalactan Proteins (AGPs) in the sexual reproduction of gymnosperms is not as well documented as that of angiosperms. In earlier studies, we demonstrated that AGPs play important roles during ovule differentiation in Larix decidua Mill. The presented results encouraged us to carry out further studies focused on the functions of these unique glycoproteins during pollen/pollen tube and ovule interactions in Larix. We identified and analyzed the localization of AGPs epitopes by JIM4, JIM8, JIM13 and LM2 antibodies (Abs) in male gametophytes and ovule tissue during pollination, the progamic phase, and after fertilization and in vitro growing pollen tubes. Our results indicated that (1) AGPs recognized by JIM4 Abs play an essential role in the interaction of male gametophytes and ovules because their appearance in ovule cells is induced by physical contact between reproductive partners; (2) after pollination, AGPs are secreted from the pollen cytoplasm into the pollen wall and contact the extracellular matrix of stigmatic tip cells followed by micropylar canal cells; (3) AGPs synthesized in nucellus cells before pollen grain germination are secreted during pollen tube growth into the extracellular matrix, where they can directly interact with male gametophytes; (4) in vitro cultured pollen tube AGPs labeled with LM2 Abs participate in the germination of pollen grain, while AGPs recognized by JIM8 Abs are essential for pollen tube tip growth