[Abnormal floral meristem development in transgenic tomato plants do not depend on the expression of genes encoding defense-related PR-proteins and antimicrobial peptides].

In this study, the morphological and cytoembryological analyses of the tomato plants transformed with the genes encoding chitin-binding proteins (ac and RS-intron-Shir) from Amaranthus caudatus L. andA. retroflexus L., respectively, as well as the gene amp2 encoding hevein-like antimicrobial peptides from Stellaria media L., have been performed. The transgenic lines were adapted to soil and grown the greenhouse. The analysis of putative transgenic tomato plants revealed several lines that did not differ phenotypically from the wild type plants and three lines with disruption in differentiation of the inflorescence shoot and the flower, as well as the fruit formation (modified plants of each line were transformed with a single gene as noted before). Abnormalities in the development of the generative organs were maintained for at least six vegetative generations. These transgenic plants were shown to be defective in the mail gametophyte formation, fertilization, and, consequently, led to parthenocarpic fruits. The detailed analysis of growing ovules in the abnormal transgenic plants showed that the replacement tissue was formed and proliferated instead of unfertilized embryo sac. The structure of the replacement tissue differed from both embryonic and endosperm tissue of the normal ovule. The formation of the replacement tissue occurred due to continuing proliferation of the endothelial cells that lost their ability for differentiation. The final step in the development of the replacement tissue was its death, which resulted in the cell lysis. The expression of the genes used was confirmed by RT-PCR in all three lines with abnormal phenotype, as well as in several lines that did not phenotypically differ from the untransformed control. This suggests that abnormalities in the organs of the generative sphere in the transgenic plants do not depend on the expression of the foreign genes that were introduced in the tomato genome. Here, we argue that agrobacterial transformation affects, directly or indirectly, expression of genes encoding for transcription factors that can activate a gene cascade responsible for the normal plant development.

[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.

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.

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.

Subcellular localization, substrate specificity and crystallization of duodenase, a potential activator of enteropeptidase.

Duodenase, a serine protease from bovine duodenum mucosa, was located in endoplasmic reticulum, the Golgi secretory granules of epithelial cells and ducts of Brunner's glands by the A-gold immunocytochemical method. Duodenase exhibits trypsin-like and chymotrypsin-like specificities with a preference for substrates having lysine at the P1 and proline at the P2 positions. The kinetic constants for the hydrolysis of 21 potential duodenase substrates are reported. The best substrates were found to be alpha-N-tosylglycylprolyllysine 4-nitroanilide (k[cat]/Km of 35000 M[-1] s[-1]), alpha-N-succinylthreonylprolyllysine 4-nitroanilide (k[cat]/Km of 18000 M[-1] s[-1]) and alpha-N-serylprolyllysine 4-nitroanilide (k[cat]/Km of 2600 m[-1] s[-1]), all of which contain the P1-P3 sequence of the enteropeptidase zymogen/activation site. On the basis of its catalytic properties and sites of localization, duodenase has been postulated to be an activator of the enteropeptidase precursor. A tetradecapeptide (LVTQEVSPKIVGGS) having the P9-P5'sequence of the cleavage site of zymogen activation of bovine proenteropeptidase was synthesized, and kinetic parameters of its hydrolysis by duodenase were determined (Km of 87 microM; k[cat] of 1.4 s[-1]; k[cat]/Km of 16000 M[-1] s[-1]). Crystals of duodenase frozen in a stream of liquid nitrogen diffracted synchrotron X-rays to 0.2-nm resolution.