ANTHERIDIAL CHROMATIN CONDENSATION FACTOR FROM MALE SEX ORGANS OF CHARA TOMENTOSA.

Incubation of roots (Melandrium noctiflorum) in crude extracts obtained from maturing male sex organs of Chara tomentosa results in supercondensation of mitotic chromosomes. The observed changes, including a number of 'secondary effects', confirm precisely with all those kinds of alterations which are typical of M-phase cells in antheridial filaments during their progression from the proliferative period of development towards terminal differentiation into sperm cells. Biological assays performed with proteinaceous extracts of maturing antheridia indicate that the activity is confined merely to a fraction containing low molecular (4.5 kDa) peptides, termed as ACCF (antheridial chromatin condensation factor). Microchemical tests showed the acidic character of ACCF and its apparent ability to form cross-links with histones and DNA-histone complexes, mediated presumably by disulfide bridges. The characteristic punctate distribution of vesicles within the cytoplasm suggests the internalization of rhodamine-isothiocyanate labelled ACCF (ACCF-TRITC) by endocytosis, implying possible sequestering and nuclear translocation upon release from the endosomal/lysosomal compartment of the cell. Copyright 1998 Academic Press.

Nucleolar patterns in antheridial filaments of mono- and dioecious Chara spp. (Characeae) with different DNA C-values.

Using NOR-silver-staining technique, nucleolar arrangements have been studied in antheridial filaments among 5 taxa of Chara with different genome sizes: monoecious Chara vulgaris L., C. fragilis Desv., C. contraria Kütz, and dioecious: Chara aspera Willd., and Chara tomentosa L. It is argued that due to a chain-series of regulatory processes establishing correlations between nucleolar cycles and consecutive cell growth cycles, the diminution of nucleoli observed throughout the development of antheridial filaments is a common and widespread feature of spermatogenesis shared by all the studied species. It is probably a controlling mechanism acting over the morphogenetic reduction of cell dimensions and cessation of mitotic divisions.

Extract from maturing antheridia of Chara induces increased condensation of mitotic chromosomes in root meristem cells.

Serving as the incubation medium for root meristems of Allium cepa and Melandrium noctiflorum, crude extracts obtained from maturing male sex organs of Chara tomentosa induce: (1) increased condensation (with both shortening and thickening) of prophase, metaphase and anaphase chromosomes, (2) disarrangement of metaphase plates and aberrant segregation of chromosomes during anaphase, and (3) abnormal decondensation of chromosomes at telophase. Numerous resemblances that exist between the natural processes occurring in male sex organs of Chara and root meristem cells from antheridial extract-treated seedlings suggest the existence of a diffusible condensation-promoting factor, responsible for the reorganization of chromatin.

Cell sizes and DNA level in antheridial filaments in relation to the initiation of spermatozoid differentiation in Chara vulgaris L.

The observations carried out indicate that the exclusion of the S phase initiation from the course of telophase of the last mitotic division in the antheridial filaments of Chara vulgaris, leading to the formation of spermatids is not a simple result of the cell size reduction, gradually accomplished in the course of the successive cell cycles (of S+G2+M type). This critical moment of spermatogenesis is probably induced by the regulators operating at the level of an antheridium. In the conditions of the long (3-5 days) darkness resulting in the cell cycle arrest in antheridial filaments at the early stage of G2 phase there is detected the operation of some additional mechanisms synchronizing spermatogenesis, which enable some retarded antheridial filaments to pass the critical control points and to enter into the process of spermiogenesis insensitive to the lack of the light. The initiation of the differentiation is accomplished either after the cell division induced by the hypothetic inductors of spermiogenesis or -- more rarely -- with omitting mitosis, i.e. in the cells containing 2C DNA.

Number, size, and transcriptional activity of nucleoli during different periods of interphase in antheridial filaments of Chara vulgaris L.

In antheridial filaments of Chara vulgaris the number of nucleoli within a single cell nucleus ranges from 3 to 12. The sizes of nucleoli vary from 0.2 to 3.5 micron in diameter. Mean number of micronucleoli, i.e. the smallest nucleoli of 0.2-0.5 micron in diameter which are distinguished after silver staining is higher than that estimated with the use of toluidine blue method according to Smetana et al. [34], the latter procedure resulting in a less contrasting visualization. Throughout the course of the whole period of interphase the mean number of nucleoli was found unchanged in successive phases and it equals some 6.5 per nucleus. Concurrently, the total volume of nucleoli increases progressively reaching maximum value by the end of the G2 phase which is attributed to the increase in number of largest nucleoli. On the basis of the analysis of 3H uridine incorporation and an in situ determination of RNA polymerase activity using the method adopted by Moore and Ringertz [25] it was evidenced that the mean transcriptional activity of nucleoli larger than 0.5 micron in diameter is not dependent upon nucleolar number within a single nucleus. It is concluded that the diverse appearance of nucleoli in cells being located precisely at the same stage of interphase reflects temporal changes of their sizes consisting in an asynchronous pulsation of individual nucleoli.

Nucleolar size and the activity of rRNA transport in the course of morphogenetic reduction of cell dimensions.

In antheridial filaments of Chara vulgaris during the first period of spermatogenesis which consists of 6 synchronous cell division cycles there occurs a gradual decrease in sizes of cells entering successive mitoses. Present studies indicate that this process is correlated with a considerable reduction of total nucleolar volume in late G2 phase which, in turn, brings about decrease in sizes of nucleoli reappearing in telophase of the subsequent cell cycle. A consequence of the above phenomenon evidenced using 3H uridine autoradiography is a gradual increase--from one generation to the next--in an amount of rRNA transported into cytoplasm due to an increase in number of small nucleoli which were found to be more active in transport than larger nucleoli. This process leads to a lowering of an increase in nucleolar volumes during consecutive interphase periods owing to a progressively limited accumulation of rRNA for the sake of daughter cells, i.e. to a spontaneously magnifying reduction of nucleolar sizes in the forthcoming cell generations. Thus, diminution of nucleoli observed during the development of antheridial filaments seems to be due to a chain-series of processes connected with mechanisms which possibly regulate rRNA transport and accompany morphogenetic events.

Changes in the activity of transport of newly synthesized RNA from nucleus and nucleolus into cytoplasm during interphase.

In antheridial filaments of Chara vulgaris given a 20 min incubation in 3H-uridine and a 40 min period of postincubation the radioactivity of extranucleolar nucleoplasm was found not altered, the radioactivity of nucleoli decreased by some 50%, and the radioactivity of cytoplasm increased by about 50%, as compared with the values obtained from preparations fixed immediately after labelling. During the periods characterized by an enhanced transcriptional activity of nucleoli, i.e. in middle stages of S and G2 phases, the most intensive RNA migration from nucleolus into the cytoplasm takes place. By the end of interphase the rate of the above process becomes slower, thus leading to accumulation of an intranucleolar pool of rRNA reserved for the sake of daughter cells.

Functional and structural heterogeneity of nucleoli: the dependence of the activity of transport of newly synthesized rRNA on nucleolar size and phase of the cell cycle.

On the basis of radioactivity of interphase cells in the antheridial filaments of Chara vulgaris after a 20 min. incubation with 3H-uridine and after a 40 min. postincubation the activity of synthesis and transport of newly synthesized RNA has been studied in different-sized nucleoli. Investigations of their ultrastructure have been also performed. It was found that the smallest nucleoli from 0.2-0.5 micron in diameter indicate the structure typical of nucleoli inactive in RNA synthesis. Nucleoli ranging from 0.5-2.0 microns in diameter which constitute the most numerous population were evidenced to be active both in synthesis and transport of newly synthesized RNA. Their structure is characterized by nucleoloneme-like appearance with small quantity of granular components. The largest nucleoli which are present in small numbers at the onset of interphase gradually increase in number till G2 phase. They indicate similar transcriptional activity as the latter category of nucleoli but a lower activity of transport of newly synthesized RNA; moreover, they abound in granular components surrounding the nucleoloneme. Nucleoli from all size-classes increase their transcriptional activity in middle S and middle G2 phases. Transport of newly synthesized RNA, however, is differentiated and it depends on both the phase of the cell cycle and nucleolar size.

DNA endoreduplication, RNA and protein synthesis during growth and development of the antheridial basal cell in Chara vulgaris L.

Cytophotometric measurements of nuclear DNA contents and morphometric analyses indicate that the level of endopolyploidy plays an important role in determining the maximum size, transcriptional and translational activity that the antheridial basal cell attains during successive stages of spermatogenesis in Chara vulgaris. During the proliferative period of antheridial development, the metabolic activity of basal cell, expressed as the total incorporation of radioactive uridine and leucine was found to increase gradually with the increasing DNA C-values, yet both the synthesis of RNA and then the synthesis of proteins become reduced at the stage preceding spermiogenesis. In accordance with some earlier data [2], the obtained results seem to support the hypothesis that regulatory mechanisms of symplasmic connections between the antheridium and a thallus participate in the regulation of morphogenesis of the male sex organs in Chara.

Ultrastructural and autoradiographic studies of non-generative cells in the antheridium of Chara vulgaris. I. Manubria.

During the development of an antheridium DNA content in manubria gradually increases to 8C-16C level. 3H-thymidine incorporation into the nuclei of the manubria lasts till the stage of quantitative predominance of the 16 celled antheridial filaments. The nucleus of the manubria is characterized by the low content of the condensed chromatin and the presence of nucleoli with the nucleolonema-like structure the number of which increases from 6-8 to 32-38 along with the increase of DNA content in a nucleus. In the cytoplasm of the manubria there are numerous secretive vesicles filled with fine-granular substance discharged outside plasmalemma, active Golgi apparatus, well-developed rough ER, numerous polysomes, mitochondria with the condensed structure and plastids with granar and inter-granar thylakoids as well as plastoglobules which increase in number and size along with the development of the antheridium. During spermiogenesis the cells are vacuolated, the number of the secretive vesicles decreases whereas the electron density of their content increases, smooth ER appears while rough ER is reduced. The manubria actively incorporate 3H-uridine, 3H-tryptophane and 3H-leucine. The increase of the incorporation activity is gradual in the period of increasing polyploidy of the manubria and rapid during the initiation of the spermatozoid differentiation. It has been suggested that the manubria should play an important role in the process of spermatogenesis and the induction of spermatozoid differentiation.

Ultrastructural and autoradiographic studies of non-generative cells in the antheridium of Chara vulgaris. II. Capitular cells.

In young antheridia, the structure of capitular cells is typical of meristematic cells. The cytoplasm is characterized by poorly developed ER system, numerous free ribosomes, active Golgi apparatus and plastids at the stage of proplastids. In the period of mitotic divisions, i.e. during formation of the initial cells of antheridial filaments, the nuclei of capitular cells have a changing structure. When capitular cells stop budding leading to the formation of successive antheridial filaments. DNA content in the nucleus is at 2C-4C level. The nucleolus with nucleolonema-like structure becomes gradually smaller in the course of the development of the anteheridium. During spermiogenesis capitular cells are vacuolated, cytoplasm contains numerous polysomes, mitochondria assume condensed structure, the incorporation of 3N-uridine and of labelled aminoacids increases. It has been suggested that capitular cells collaborate with other antheridial cells in the regulation of the course of spermiogenesis.

Cell cycle arrest in antheridial extract-treated root meristems of Allium cepa and Melandrium noctiflorum.

Previous results have demonstrated that extracts derived from maturing male sex organs of Chara tomentosa are capable of inducing profound structural and functional effects upon M-phase cells in the primary root meristems of Melandrium noctiflorum and Allium cepa. Evident changes produced by a putative factor engaged in morphogenesis of antheridial filaments are manifested by: (1) significant shortening of chromosomes, (2) decreased mitotic indices, and (3) altered proportions estimated for the prophase and telophase transit times. The present image analysis of late G2 phase nuclei in antheridial filaments of C. tomentosa supports the concepts that progressive changes of their functional activities correspond closely to the increasing proportion of condensed chromatin. Cytophotometric measurements of Feulgen-stained cell nuclei in root meristems after a prolonged incubation in antheridial extracts revealed that cells which previously divided asynchronously became preferentially arrested in G1 (M. noctiflorum) and G2 (A. cepa). The stages at which the cells arrest are supposed to counterpart restriction checkpoints that prevent the initiation of DNA synthesis and mitosis. This assumption has been confirmed by autoradiographic studies using 3H-thymidine. In terms of the "Principal Control Points" (PCP) hypothesis, the obtained results suggest that two PCPs regulate G1-->S and G2-->M transition in a nuclear structure-dependent and a species-specific manner. Although in antheridial extract-treated roots of both M. noctiflorum and A. cepa there are only slight changes in the levels of chromatin condensation, the relative proportions of G1- and G2-arrested cells and their nuclear density profiles differ, as compared with the control and carbohydrate-starved plants.

Effects of antheridial peptides derived from Chara on cell cycle-regulated processes in human and yeast cells.

Previous experiments have shown that extracts obtained from maturing male sex organs of Chara tomentosa, containing a low molecular weight peptide (termed antheridial chromatin condensation factor--ACCF) are capable to induce structural and functional effects in root meristems and fern gametophytes. Our present data point to a number of resemblances between the phenotypic characters of antheridial filaments (a.f.) and the properties of ACCF-treated human lymphocytes and HeLa cells; these comprise primarily a number of morphological changes at the nuclear/chromosomal levels. Mitotic chromosomes become shortened and the relative duration of prophase is reduced, while duration of telophase is prolonged. Nucleolar profiles in ACCF-treated HeLa cells become reduced. Significant decrease in mitotic activity was found in human and yeast cells (Schizosaccharomyces pombe). All the above similarities between the "innate" processes within a.f. and those induced by ACCF provide positive evidence for the presence of a highly specific factor that contributes to nuclear re-patterning of cells undergoing morphogenetic transformations before the onset of spermiogenesis.

The impact of copper ions on growth, lipid peroxidation, and phenolic compound accumulation and localization in lentil (Lens culinaris Medic.) seedlings.

Changes in phenolics (PhC) measured as UV-absorbing compounds (UVAC) and their localization as well as growth, lipid peroxidation (TBARS level), H(2)O(2) and Cu accumulation and other ions content in roots of two lentil cultivars (cv. Krak and cv. Tina) contrasting in Cu sensitivity were examined. The aim of this study was to reveal the importance of PhC in Cu tolerance. During Cu treatment, inhibition of growth and increases in lipid peroxidation in roots of both cultivars were observed, but the effects were more pronounced in cv. Tina (more sensitive) than in cv. Krak (less sensitive). Cu at 0.5 mM caused higher Cu and H(2)O(2) accumulation, but lower K(+) content and UVAC levels in the root tips of cv. Tina. Opposite changes were recorded in cv. Krak. Fluorescence microscopic analyses confirmed greater PhC accumulation in cv. Krak (less sensitive) than in cv. Tina (more sensitive) after Cu treatment and showed that these compounds accumulated particularly in vacuoles and the cell wall. Taken together, these results show that, in spite of the high concentration of Cu-stimulated PhC accumulation in cv. Krak, it was not sufficient to counteract the amount of ROS generated by the metal. The role of PhC in different reactions to Cu stress in lentil roots is discussed.

Changes in ultrastructure of plasmodesmata during spermatogenesis in Chara vulgaris L.

Two types of plasmodesmata are found within an antheridium of Chara vulgaris: open plasmodesmata filled with electron-transparent cytoplasm, and plugged plasmodesmata, filled with an osmiophilic dense substance. Open plasmodesmata occur only between cells synchronized completely in respect of their advancement in cell-cycle progression or differentiation. Plugged plasmodesmata connect different types of cells or cells of the same type at various stages of the cell cycle. Open plasmodesmata may become plugged, and vice versa. These changes are connected with the limitation or extension of synchronization of cellular divisions and differentiation within the groups of cells in the antheridium.

Heterogeneity of the early G2 phase-arrested cells; cytophotometric, interferometric, and EM morphometric studies.

Cells of antheridial filaments in Chara vulgaris L. blocked at the early G2 phase by a 5 day dark treatment were studied cytologically in order to determine causes of their heterogeneous response to light-induced mitotic activation. Cell size distribution patterns in dark inhibited populations were shown to be nearly consistent with those found within the control populations, comprising all the stages of interphase; cells which resume divisions in response to photoinduction attain, however, sizes characteristic of mitosis occurring in natural light conditions. Cytophotometric determinations of nuclei in cells given dark treatment indicated a complete homogeneity of populations in respect of replicated DNA contents. As evidence by nucleus-cytoplasm surface relations, interferometric measurements of their dry mass contents, and EM stereometric studies, the heterogeneity of early G2 phase arrested populations results from an imbalance between nuclear DNA-division cycle, which is continued up to the early stage of the G2 period, and the growth cycle, which in turn, due to its pronounced inhibition gives rise to the deficit of cytoplasm. Since no shortages in total volumes of structural cytoplasm components were found the number and volume of mitochondria were shown even largely increased, it is assumed that asynchrony during light-induced mitotic reactivation originates from quantitative differences of constituents within the area of ground cytoplasm.

Changes in the activity of RNA polymerase detected in situ and the intensity of 3H uridine incorporation into the nucleolus and the nucleus of interphase cells in antheridial filaments of Chara vulgaris L.

Analogously as in the 32-cell stage [20], in the 4-, and 8-cell generations of antheridial filaments of Chara vulgaris, the activity of DNA dependent RNA polymerase detected in situ as well as the 3H uridine incorporation increase in the middle S phase and in the middle G2 phase, while they decrease considerably at the end of S phase and in late G2 phase. The diphasic changes occur both in the nucleolar and extranucleolar (nucleoplasmic) activity of RNA polymerase as well as in the 3H uridine incorporation. However, the maximum nucleolar activity, in both S and G2 phases, precedes the peak of nucleoplasmic activity. During the increased nucleolar activity RNA polymerase (as calculated per nucleus) shows a higher level as compared with nucleoplasmic RNA polymerase, whereas the intensity of 3H uridine incorporation into nucleous and nucleoplasm is similar. It may be supposed that the incubation environment containing Mg2+ used in vitro is more stimulating for the nucleolar RNA polymerase than for the nucleoplasmic RNA polymerase. The mean transcriptional activity of the nucleus and the activity of RNA polymerase in the 8-cell generation is about 20% lower than in the 4-cell generation, in proportion to the decrease in cell sizes.

Transport of glutathione S-conjugates in the yeasts Saccharomyces cerevisiae.

Transport of 2,4-dinitrophenyl-S-glutathione (DNP-SG) and a fluorescent glutathione S-conjugate, bimane-S-glutathione (B-SG) was studied in the baker's yeasts (S. cerevisiae). Both conjugates were exported from the cells; the transport was inhibited by fluoride and vanadate like in mammalian cells. B-SG was also found to be accumulated in the vacuoles. The transport rate of DNP-SG outside the cell was higher in a vacuolar-deficient strain. A significant ATP-dependent uptake of (3H)-DNP-SG by vacuoles was found. These results indicate that S. cerevisiae transport glutathione S-conjugates both outside the cells and into the vacuoles.