Structure of antipodal cells nuclei of wheat embryo sac during programmed cell death.

MAIN CONCLUSION: During antipodal cells PCD, polytene chromosomes rearrangement, segregation of nucleoli components and extrusion of nuclear components occur, cytochrome c is released from the mitochondria and DNA breaks appear. We studied in detail the nuclei of cells of the antipodal complex of wheat embryo sac (Triticum aestivum L.) during programmed cell death (PCD). The antipodal complex has been reported to be formed before double fertilisation of the embryo sac. Polyploidisation leads to the formation of giant polytene chromosomes in the nuclei of antipodal cells. These chromosomes are involved in secretory functions and are important for the development of cellular endosperm. Terminal deoxynucleotidyl transferase dUTP nick end labelling assay and immunodetection revealed DNA breaks in the nuclei and release of cytochrome c from mitochondria into the cytoplasm of antipodal cells during PCD. We used transmission electron microscopy, immunodetection and histochemistry to analyse the characteristic structural changes in the nuclei of antipodal cells during PCD. These included sequential structural changes in the nuclei containing polytene chromosomes, segregation of some components of the nucleolus into the bodies of polytene chromosomes, extrusion of nucleolar components and parts of chromosomes into the cytoplasm of antipodal cells and then into the endosperm coenocyte. The obtained results expand the understanding of the structural changes of plant cells with giant polytene chromosomes during PCD.

REORGANIZATION OF INTERPHASE MICROTUBULES IN ROOT CELLS OF MEDICAGO SATIVA L. DURING ACCLIMATION TO OSMOTIC AND SALT STRESS CONDITION.

We have investigated the organization of microtubule system in interphase cells of Medicago sativa L. roots during acclimation to salt and osmotic stress at different concentrations of NaCl, Na2SO4 and mannitol. We have identified several morphological changes in tubulin cytoskeleton that appear during the acclimation to salt and osmotic stress in the cells of different root tissues: 1) decreased density of cortical microtubule network, 2) random orientation of cortical microtubule bundles, 3) non-uniform density of the bundles, 4) thickening of the bundles, 5) fragmentation of the bundles, 6) formation of centers of converging microtubule. Reduced density of the microtubule network and thickening of the bundles was detected during osmotic and salt stress, yet random orientation of cortical microtubules was observed under osmotic stress and not found during salt stress. Fragmentation of microtubule bundles was apparent during salt stress and less evident at high concentration of mannitol. Formation of centers of converging microtubule was common under prolonged action of sodium sulfate, less common under sodium chloride and not found after mannitol treatment. Our data show that cortical microtubules in alfalfa root cells rearrange not only in response to different ions, but also to osmotic pressure. Thus, the signaling pathways and molecular mechanisms inducing reorganization of the microtubule system may be triggered not only by sodium cations but also by sulfate and chloride anion at the concentrations that do not cause irreversible cell damage. Our study show that the osmotic and salt stress differently affect the cortical microtubules, and their reorganization in response to stress depends on the salt cations as well as anions might also show additional effect under salt stress.

[Antipodal complex development in the embryo sac of wheat].

Dynamics of an antipodal complex formation in wheat (Triticum aestivum L.) has been observed in detail using a reconstruction of serial semifine sections. Three consecutive crucial stages have been identified in the development of the antipodal complex: (1) proliferation of initial cells, (2) growth and functional differentiation of antipodal cells, and (3) cell apoptosis. Specific features of the mitotic division of antipodal cells have been characterized. It has been shown that the structure of interphase nuclei and mitotic chromosomes of proliferating antipodal cells is similar to that of nucellar cells surrounding the embryo sac. According to the reconstruction of appropriately oriented serial sections, the division of antipodal cells is asynchronous. DNA content in differentiated antipodal cells has been determined by a cytophotometric analysis; in the case of a mature embryo sac, the ploidy of antipodal cells varied from 8 to 32C. Proliferation and DNA endoreduplication processes in the antipodal complex proceed at different time; the second process starts only after the termination of the first one. DNA endoreduplication is accompanied by total chromatin remodeling; as a result, giant chromosomes are formed in the nuclei of antipodal cells. The final stage of the antipodal complex development is programmed cell death or apoptosis. A model for the structural organization of an antipodal complex has been proposed based on the layer arrangement of cells. The secretory activity of antipodal cells directed towards the endosperm syncytium has been detected for the first time. The analysis of "truncated" ovules with an undeveloped endosperm has shown that developing endosperm can be a possible inductor, which stimulates the functional activity of antipodal cells and triggers their terminal differentiation. The obtained results evidence the functional role of antipodal cells in the development of the endosperm and embryo.

Multi-walled Сarbon Nanotubes Penetrate into Plant Cells and Affect the Growth of Onobrychis arenaria Seedlings.

Engineered nanoparticles (ENPs) are now being used in many sectors of industry; however, the impact of ENPs on the environment still requires further study, since their use, recycling, and accidental spill can result in the accumulation of nanoparticles in the atmosphere, soil, and water. Plants are an integral part of ecosystems; hence their interaction with ENPs is inevitable. It is important to understand the consequences of this interaction and assess its potential effects. The present research is focused on studying the effects of the industrial material Taunit, containing multi-walled carbon nanotubes (MWNTs), on plants, and testing of its ability to penetrate into plant cells and tissues. Taunit has been found to stimulate the growth of roots and stems and cause an increase in peroxidase activity inOnobrychis arenariaseedlings. Peroxidase activity increases with decreasing concentration of Taunit from 1,000 to 100 mg/l. MWNTs from Taunit were detected in the cells and tissues of seedling roots and leaves, implying the ability of MWNTs to penetrate into roots and accumulate there, as well as their ability to be transported into seedling leaves. Thus, the changes in the physiological parameters of plants are associated not only with MWNT adsorption on the root surface, as previously believed, but also with their penetration, uptake and accumulation in the plant cells and tissues.

New isoform HvNHX3 of vacuolar Na+/H+-antiporter in barley: expression and immunolocalization.

The gene HvNHX3 encoding a new isoform of vacuolar Na+/H+-antiporter was identified in barley. This gene is expressed in roots and leaves of barley seedlings, and it encodes a protein consisting of 541 amino acid residues with predicted molecular weight 59.7 kDa. It was found that by its amino acid sequence HvNHX3 is closest to the Na+/H+-antiporter HbNHX1 of wild type from Hordeum brevisibulatum that grows on salt-marsh (solonchak) soils (95% homology). The expression of HvNHX3 during salt stress is increased several-fold in roots and leaves of barley seedlings. At the same time, the amount of HvNHX3 protein in roots does not change, but in leaves it increases significantly. It was shown using HvNHX3 immunolocalization in roots that this protein is present in all tissues, but in control plants it was clustered and in experimental plants after salt stress it was visualized as small granules. It has been proposed that HvNHX3 is converted into active form during declusterization. The conversion of HvNHX3 into its active form along with its quantitative increase in leaves during salt stress activates Na+/H+-exchange across the vacuolar membrane and Na+ release from cytoplasm, and, as a consequence, an increase of salt stress tolerance.

[The effect of low temperature on the microtubules in root meristem cells of spring and winter cultivars of wheat Triticum aestivum L].

We have studied the response of the interphase and mitotis microtubule arrays in root meristem cells of spring and winter cultivars of wheat Triticum aestivum L. (Moskovskaya 35 and Moskovskaya 39) during cold stress (1 h at 0 degrees C) and acclimation to cold (3-48 h at 0 degrees C). Our data show that interphase microtubules are more resistant to cold than mitotic arrays in both cultivars. During cold stress the density of endoplasmic microtubules increases in interphase cells of winter plants, yet no changes are detected in cells of spring plants. In mitotic cells of both wheat cultivars the density of microtubules within the kinetochore fibers decreases, yet this effect is more evident in the cells of spring plants. During acclimation to cold of both cultivars, we have observed the disorganization of the interphase cortical arrays and the enhanced growth of endoplasmic microtubule arrays, composed of microtubule converging centers. However, the reaction of mitotic microtubule arrays differs in the cells of winter and spring plants. In winter plants, during prophase diffuse tubulin "halo" accumulates first at perinuclear area, followed by the appearance of the microtubule converging centers. In spring plants, we have observed the formation of the prophase spindle, yet later the prophase spindle is not detected. Metaphase cells of both cultivars show similar aberrations of the mitotic spindle, accumulation of abnormal metaphases and the excessive formation of microtubule converging centers. In telophase cells of both cultivars, acclimation induces similar reaction, resulting in the disorganization of the phragmoplast and the formation of multiple microtubule converging centers. The latter are detected in the perinuclear areas of the daughter cells in winter plants and in the cortical cytoplasm of cells in spring plants. Our data point to the common pathways of microtubule response to cold treatment (0 degrees C). The excessive formation of the microtubule converging centers indicates the activation of microtubule assembly during prolonged cold treatment.

[High molecular weight protein detected in higher plant cells by antibodies against dynein is associated with vesicular organelles including Golgi apparatus].

The cytoplasmic dynein is a multisubunit complex driving organelles along microtubules to their minus-end. We used antibodies against two functional domains (motor and microtubule-binding) of one of principal components of the complex--dynein heavy chain of slime mould Dictyostelium discoideum--to test root meristem cells of wheat Triticum aestivum. The antibodies reacted with a high molecular weight protein (> 500 kDa) in the total cell extract and the band recognized by the antibodies in plant extracts had a lower electrophoretic mobility than the high molecular weight band of mammalian dynein. Antibodies coupled to protein A-Sepharose precipitated the high molecular weight protein from the purified cell extracts. Immunocytochemical analysis demonstrated that the antigen recognized by antibodies against dynein heavy chains is associated with the vesicles whose localization depends on the cell cycle stage. The antigen-positive vesicles were localized to the perinuclear region in interphase and early prophase, to the spindle periphery and to spindle pole region during mitosis, and to the interzonal region in the period of fragmoplast and cell plate formation. Some antigen-positive vesicles also reacted with antibodies against Golgi protein markers. The obtained data indicate that higher plant cells contain a high molecular weight protein interacting with antibodies against the motor and microtubules-binding domains of Dictyostelium dynein heavy chain. The revealed antigen was associated with the vesicular structures in the cytoplasm including the Golgi apparatus.

[Localization of fibrillarin, 53 kDa protein and Ag-NOR proteins in the nuclei of giant antipodal cells of the wheat Triticum aestivum]. / Osobennosti lokalizatsii fibrillarina, belka c mol. massoi 53 kDa i Ag-NOR-belkov v gigantskikh iadrakh kletok antipod pshenitsy.

Distribution of nucleolar argentophylic proteins, fibrillarin and 53 kDa protein, in highly polyploid nuclei of antipodal cells of Triticum aestivum L. was studied at different stages of the embryo sac development. The main results are as follows. 1. Ag-NOR proteins and fibrillarin form clusters are distributed in the giant nucleoli, whereas 53 kDa protein is mainly localized on the nucleolar periphery. Ag-NOR proteins and fibrillarin are accumulated as globular nucleolar-like particles--micronucleoli. 2. Dynamics of Ag-NOR proteins, fibrillarin and 53 kDa protein depends on the proliferative activity of endosperm cells. In embryo sacs with non-dividing endosperm cells at interphase stages, Ag-NOR proteins and fibrillarin were observed only within nucleoli and micronucleoli. In embryo sacs with dividing endosperm cells, fibrillarin and 53 kDa protein formed heterogeneous globular bodies varying in size. Simultaneously, some argentophylic material was observed in giant chromosomes. This may be due, presumably, to a partial or complete disappearance of the nucleoli of antipods and transition of some nucleolar components into the peripheral material of giant polytene chromosomes. We suggest that giant nuclei of antipodal cells may undergo cyclic transformation similar to those in the nuclei of dividing cells.

Activation of latent nucleolus organizers induced by experimental polyploidization in cells of hexaploid wheat Triticum aestivum L.

The effect of prolonged colchicine-induced polyploidization on activation of latent nucleolus-organizing regions (NOR) of chromosomes was studied in diploid meristematic cells and polyploid root cells of Triticum aestivum L. It has been shown that control diploid and tetraploid cells have maximal number of nucleoli equal to four, which corresponds to the number of nucleolar chromosomes (NC) with active (visualized by staining with AgNO3) NOR (two pairs of homologous chromosomes 1B and 6B). Treatment of wheat seedlings with colchicine for 30 h results in following changes in polyploid cells: (1) impregnation of NOR with silver is observed on homologues of either chromosomes 1A or 5D in all tetraploid metaphase plates (4n, 2x, 4c), which is indicative of the NOR activation on this chromosome in pre-mitotic polyploid interphase; (2) In tetraploid metaphase, NOR in all four homologues of activated chromosomes or in only two of them may be stained; (3) maximal number of nucleoli in tetraploid nuclei is increased till 12, which confirms activation of transcription of additional rRNA gene clusters in polyploids; (4) activation of the rRNA gene expression is induced by the cell polyploidization rather than by colchicine, since in the colchicine-treated diploid cells both maximal number of nucleoli and the number of metaphase chromosomes with active NOR is not changed as compared with control. The obtained data allow us to suggest that structural "separation" of NC in polyploid nuclei stimulates activation of latent NOR.

[Effect of fusococcin on the genome of common wheat]. / Deistvie fuzikoktsina na genom miagkoi pshenitsy.

The effect of fusicoccin on the common wheat genome (cv. Mironovskaya 808) during early phases of seed germination was studied. It was shown that fusicoccin: (1) increased the number of cells with one or two nucleoli and decreased the proportion of cells containing three and four nucleoli; (2) enhanced the total volume of nucleoli per nucleus at a concentration of 0.68 mg/l; (3) did not activate additional rRNA genes on chromosomes 1B and 6B; (4) did not activate the latent nucleolus organizer regions on chromosomes of genome B; (5) induced associations of nucleolus organizer regions on B-genome chromosomes; (6) significantly enhanced mitotic activity in apical meristem of seedling root tips and promoted the first peak of mitoses; the effect disappeared after 44 h of seed imbibition; (7) in contrast to gibberellin, did not synchronize cell division; (8) did not cause chromosome and chromatid aberrations and increase the frequency of sister chromatid exchanges at physiologically active concentrations. Presumable mechanisms of fusicoccin-induced activation of rRNA genes are discussed in relation to the identification of the fusicoccin receptor belonging to the GF14 proteins with an intracellular binding site, to the detection of endogenous fusicoccin in plants in vivo, and to fusicoccin-induced activation of gene transcription.

Dynamics of structural and functional association of nucleolar chromosomes in cells of the hexaploid wheat Triticum aestivum L. at different stages of the cell cycle and during genome polyploidization.

Quantitative analysis of interphase association of the nucleolar chromosomes at different stages of the cell cycle and during genome polyploidization was carried out. Cells of various tissues of hexaploid wheat Triticum aestivum L. (Moskovskaya-35) were used, including diploid root meristematic cells, endopolyploid root cells, triploid endosperm cells and antipodal cells with polytene chromosomes. Interphase nucleoli impregnated with silver or stained with autoimmune antibodies to 53 kDa nucleolar protein served as markers of the nucleolar chromosome association. The following data were obtained: (1) silver-staining revealed two pairs of homologous chromosomes 1B and 6B with active nucleolus-organizing regions in the root meristematic cells; (2) maximal number of nucleoli in diploid meristematic cells reaches four, which corresponds to the number of chromosomes with active organizers; (3) analysis of cells at different stages of the cell cycle has shown that the tendency to the nucleoli association is observed as soon as cells pass individual stages of the cycle; (4) after DNA and chromosome reduplication, the nucleolus-organizing regions in sister chromatids function as a common structure-functional complex; (5) in endopolyploid root cells and antipodal cells with polytene chromosomes, the number of nucleoli does not correlate with ploidy level, and an additional nucleolus revealed in some cells is the result of activation of the latent organizer in one of the nucleolar chromosomes; (6) in the triploid endosperm nucleologenesis, the stage of prenucleolar bodies is missing. Our data suggest that "fusion" of nucleoli and reduction of their number due to the "satellite" association of the nucleolar chromosomes are two independent processes regulated by different mechanisms.