Nuclear fusions contribute to polyploidization of the gigantic nuclei in the chalazal endosperm of<Emphasis Type="Italic"> Arabidopsis</Emphasis>

Somatic polyploidization is recognized as a means to increase gene expression levels in highly active metabolic cells. The most common mechanisms are endoreplication, endomitosis and cell fusion. In animals and plants the nuclei of multinucleate cells are usually prevented from fusing. Here, we report that the nuclei from the syncytial cyst of the chalazal endosperm of Arabidopsis thaliana (L.) Heynh. are polyploid with some intermediate ploidy levels that cannot be attributed to endoreplication, suggesting nuclear fusion. Analysis of isolated nuclei, together with fluorescent in situ hybridization (FISH), revealed that nuclei from the chalazal endosperm are two or three times bigger than the nuclei from the peripheral endosperm and have a corresponding increase in ploidy. Together with the consistent observation of adjoined nuclei, we propose that nuclear fusion contributes, at least in part, to the process of polyploidization in the chalazal endosperm. Confocal analysis of intact seeds further suggested that free nuclei from the peripheral endosperm get incorporated into the chalazal cyst and likely participate in nuclear fusions.Abbreviations BAC Bacterial artificial chromosome - CZE Chalazal endosperm - DAPI 4,6-Diamino-2-phenylindole - FISH Fluorescent in situ hybridization - NOR Nucleolar organizing region - NCD Nuclear cytoplasmic domain - PEN Peripheral endosperm     

Analysis of chromatin and DNA during chromosome endoreduplication in the endosperm ofTriticum durum Desf.

Summary Chromatin structure was studied in nuclei of the endosperm of durum wheat (Triticum durum Desf., cv. Creso), where a large number of cells undergo chromosome endoreduplication during caryopsis development. Optical density profiles of interphase nuclei at different ploidy levels after Feulgen staining were determined cytophotometrically. It was observed that, within each development stage, polyploid nuclei (6–12C and 12–24C) show more condensed chromatin than euploid nuclei (3–6C): this should indicate that endoreduplication is accompanied by some reduction of nuclear activity. Within the same ploidy level, 3–6C and 6–12C nuclei become increasingly condensed with development (except for the last stage), while 12-24C nuclei are identical at all stages. DNA methylation at different stages of caryopsis development was then analyzed in genomic DNA, highly repeated sequences and ribosomal DNA, by digestion with cytosine-methylation-sensitive restriction enzymes. We observed that (i), depending on the enzyme, DNA from caryopses may show higher mean length than DNA from shoot apices and variations occur during endosperm development; (ii) highly repeated DNA sequences also show some variation in base methylation between apices and endosperms and among endosperm development stages, even though to a lesser extent than genomic DNA; (iii) rDNA shows variations only between endosperm and apices while no variation was observed among endosperm development stages in relation to chromosome endoreduplication. Our data may be explained by assuming the occurrence, during endosperm development, of processes of chromatin condensation possibly involved in silencing the activity of extra copies of DNA resulting from chromosome endoreduplication. At least in part, DNA methylation is involved in the process of chromatin condensation. rDNA shows no variation during endosperm development: this suggests that rDNA copies are actively transcribed in both triploid and endoreduplicated nuclei.     

Unexpected DNA content in the endosperm of Cupressus dupreziana A. Camus seeds and its implications in the reproductive process

 Nuclear DNA content of embryo and endosperm from mature and immature Cupressus dupreziana A. Camus seeds was estimated using laser flow cytometry. Relative DNA content of endosperm nuclei corresponded to four ploidy levels: 2C, 4C, 6C and 8C. The embryo nuclei invariably exhibited a diploid pattern. In all endosperm tissue analyzed no haploid nucleus was found. This is problematic since, in gymnosperms, endosperm and female gametes originate from one functional haploid megaspore produced by meiosis. The possible origin and derivation of C. dupreziana endosperm are discussed in light of previous results concerning the two other Mediterranean cypresses, C. sempervirens and C. atlantica. Received: 15 January 1998 / Revision accepted: 27 March 1998     

Developmental events in ovules of the ornamental plant Rudbeckia bicolor Nutt

Microscopic observations of R. bicolor ovules showed that tetrasporic embryo sacs of Fritillaria type are formed. In the mature female gametophytes modifications in antipodal cell formation and egg apparatus organization were observed, e.g. morphological resemblance was evident of antipode or synergid to the egg cell. In the central cell cytoplasm of the mature gametophytes the presence of small bodies was a characteristic feature. Development of both embryo and endosperm was observed in ∼73% of ovules at the embryo stage, while retarded or arrested development of the endosperm was found in ∼26% of them. Occasionally, two embryos occurred in the embryo sac. This is the first record of polyembryony in this species. Although hemigamy has been previously described in Rudbeckia bicolor Nutt., in the present investigations mosaic structure of embryos was not detected. Measurements of the C-DNA amount (flow cytometry) revealed embryo nuclei with 2C DNA content only, and endosperm nuclei with 5C DNA content in the mature seeds. No peak corresponding to 1C nuclei was detected in the histograms obtained from the nuclear preparation of seeds or seedling parts. These results suggest that hemigamy is not an obligatory phenomenon in R. bicolor. The mean 2C DNA value was determined as 14.51 pg (the first estimation for this species).     

Morphological and cytofluorometric study of the giant cells of the trophoblast of the common vole]

The primary and secondary giant cells of trophoblast in placenta Microtus arvalis were studied. The giant polyploid nuclei are formed in result of series of successively proceeding endomitotic polyploidization of chromosomes. Two stages of endomitosis are described: endointerphase with the uniform net of thin chromatin threads and the stage when small round or rod-shaped paired chromosomes gather mostly under the nuclear membrane. Great number of round, oval, and complex-shaped nucleoli may be seen in nuclei during both stages of endomitosis, the number growing during polyploidization. The morphology of the chromosome-nucleolar apparatus involves peculiarities of the polyploidization mechanism in placenta Microtus arvalis trophoblast. Endomitosis occurs both in low and high-polyploid nuclei. Cytofluorometric determination of the DNA amount in nuclei polyploid nature. The degree of polyploidy of the trophoblast giant cells nuclei during terminal differentiation of placenta corresponds to 128c-512c, and some nuclei contain the DNA amount corresponding to 1024 and 2048 chromosomal sets. The cause of origin of the polyploid cells in trophoblast of rodents placenta is discussed.     

Human hepatocyte polyploidization kinetics in the course of life cycle

The processes of polyploidization in normal human liver parenchyma from 155 individuals aged between 1 day and 92 years were investigated by Feulgen-DNA cytophotometry. It was shown that polyploid hepatocytes appear in individuals from 1 to 5 years old. Up to the age of 50 years the accumulation rate of binucleate and polyploid cells is very slow, but subsequently hepatocyte polyploidization is intensified, and in patients aged 86–92 years the relative number of cells with polyploid nuclei is about 27%. Only a few hepatocytes in the normal human liver reach 16C and 8C×2 ploidy levels for mononucleate and binucleate cells respectively. Using a mathematical modeling method, it was shown that during postnatal liver growth the polyploidization process in human liver is similar to that in the rat, and that polyploid cells are formed mainly from binucleate cells. As in rats, prior to an increase in ploidy level, diploid human hepatocytes can pass several times through the usual mitotic cycles maintaining their initial ploidy level. After birth, only one in ten hepatocytes starting DNA synthesis enters the polyploidization process. At maturity about 60% of 2C-hepatocytes starting DNA synthesis divide by conventional mitosis, the rest dividing by acytokinetic mitosis leading to the formation of binucleate cells. During ageing the probability of hepatocyte polyploidization increases and in this period there are two polyploid or binucleate cells for every diploid dividing by conventional mitosis.     

Aging of chick embryo fibroblasts in vitro : III. Polyploid cell accumulation

The accumulation of polyploid cells during the lifespan of chick embryo fibroblasts was examined. The nuclear area of each stained nucleus in the chick cells is virtually proportional to its relative DNA content. Changes in mean nuclear area of the cells with advancing aging were observed. The mean nuclear area increases at the latest culture stage when growth rate notably declines, and their increase follows an increase in multinuclear cells. In the senescent cells, there are the ploidy classes of 2C, 4C, 8C, 16C, 32C and 64C, being defined as 2ⁿC. The mechanism of age-dependent polyploidization is also discussed.     

Both Nucleolar Organizers Are Replicated in Dipteran Polyploid Tissues: A Study at the Level of Individual Nuclei

Working with the Dipteran Calliphora erythrocephala, we have tested the hypothesis that only one nucleolar organizer region (NO) is replicated during polyploidization. NO replication was examined in two very different highly polyploid nuclear types: salivary gland nuclei and nurse cell nuclei. Two strains of the organism containing NO regions with highly diagnostic nontranscribed spacer (NTS) polymorphisms were prepared and reciprocal single pair-matings between members of the strains were performed. The representation of the two distinguishable NOs in diploid and polyploid DNAs of individual F1 progeny from each cross was then examined. DNA from a total polyploid nuclear DNA preparation and from individual polyploid nuclei of both tissue types was analyzed. Our results show conclusively that both genomic NOs are replicated in individual polyploid nuclei of both types. Further, evidence for variation in the relative replication of cistrons from the two NOs by individual nuclei was obtained. The cistron types present in the NOs of both strains showed differential replication upon polyploidization. In general, the patterns of differential cistron replication seen in salivary gland and nurse cell nuclei were similar.     

Chromosome organization in wheat endosperm and embryo

We have analysed the chromosome organization in endosperm and embryo of bread wheat (Triticum aestivum L.), in order to compare these tissues with developing anthers, in which the centromeres associate, and the developing root xylem vessel cells, in which the chromosomes endoreduplicate to become polytene and associate via their centromeres. Both endosperm and embryo showed a typical Rabl configuration and a degree of non-homologous centromere association and the endosperm also showed extensive telomere association. Wheat endosperm is initially triploid and during its development a percentage of the nuclei increase their DNA content to 6C and 12C. 6C nuclei showed twice as many centromeres as 3C nuclei and the centromere number increased further in 12C nuclei. The higher the C-content of a nucleus the more the telomeres associated in endosperm. The vast majority of 12C nuclei showed six rye chromosome arms, although a few showed three associated groups of rye chromosome arms. This means that during endosperm development wheat nuclei show both polyploidization and polytenization.     

Microtubules in early development of the megagametophyte of <Emphasis Type="Italic">Ginkgo biloba</Emphasis>

Food storage tissue in the seeds of gymnosperms is female gametophyte (megagametophyte) that develops before fertilization, whereas, in seeds of angiosperms, food is stored as endosperm initiated by double fertilization. The megagametophyte is haploid, and endosperm is usually triploid, at least initially. Despite differences in origin, ploidy level, and developmental trigger, the early events of female gametophyte development in ginkgo are very similar to nuclear endosperm development in the seeds of angiosperms. In both, development begins as a single cell that undergoes multiple mitoses without cytokinesis, to produce a large syncytium. This study provided evidence that microtubule involvement in organization of the syncytium into nuclear cytoplasmic domains (NCDs) via nuclear-based radial microtubule systems is a critical developmental feature in the ginkgo megagametophyte, as it is in endosperm. Once the initial anticlinal walls have been deposited at the boundaries of NCDs, cellularization proceeds by the process of alveolation. Continued unidirectional growth of the alveolar walls is an outstanding example of polar cytokinesis. Ginkgo megagametophyte development appears to occur uniformly throughout the entire chamber, whereas nuclear type endosperm usually exhibits distinct developmental domains. These observations suggest that there is a fundamental pathway for the development and cellularization of syncytia in seed development.     

Flow cytometric analysis of polysomaty and in vitro genetic instability in potato

The analysis of nuclear DNA contents in various tissues of potato genotypes showed that flow cytometry is a rapid method to characterize large populations of cells for polysomaty, that is, the occurrence of cells with normal DNA levels together with cells containing endoreduplicated nuclei. The proportion of endoreduplicated nuclei varied in different tissues and genotypes of potato. The analysis of callus and cell cultures showed that the temporal changes in nuclear DNA contents during in vitro growth can be followed and the degree of polyploidization quantified. It is concluded that flow cytometry is a highly suitable method to detect ploidy changes in differentiated plant tissues and calli which are often not amenable for chromosome number determination.     

Follicle cell polyploidy in Leucophaea maderae: Regulation by juvenile hormone

Microspectrophotometric analysis of Feulgen-stained nuclei of the terminal follicle cells in the cockroach Leucophaea maderae showed that during maturation the follicle cells became polyploid. In virgin females, the follicle cell nuclei were diploid. After mating, and during vitellogenesis, the ploidy of the follicle cells increased from 2 C to 32 C with a small percentage of 64 C nuclei. There was no further increase in the ploidy levels during the chorionic stage of development.Injections of juvenile hormone III into decapitated virgin females elevated the ploidy levels in the follicle cells. The DNA content of these nuclei at 96–120 h after injection of juvenile hormone III increased from 2 C to 4 C. Such polyploidization of nuclei was dose-dependent with the highest DNA content occurring in response to 25–50 μg juvenile hormone III. The juvenile hormone-induced increase in DNA content correlated with an increase in the rate of [³H]thymidine incorporation into DNA.Our data suggest that the role of juvenile hormone in follicle cell development during the vitellogenic period, whether direct or indirect, is to promote selectively a large increase in the DNA content of the cells. This may facilitate the next stage of follicle cell development, choriogenesis.     

DEVELOPMENT OF THE SEED OF SOLANUM PHUREJA

Dnyansagab , Vishnu R., and Delmer C. Cooper . (U. Wisconsin, Madison.) Development of the seed of Solanum phureja. Amer. Jour. Bot. 47(3) : 176—186. Illus. 1960.—Ontogeny of the seed of Solanum phureja Juz. et Buk. is described. The megagametophyte, during the course of its development, ruptures the nucellus and at maturity lies in direct contact with the inner layer (endothelium) of the single massive integument. The mature megagametophyte, a 7-celled structure, consists of a 3-celled egg apparatus, an endosperm mother cell with fused polar nuclei and 3 persistent antipodals. Both 2- and 3-celled mature pollen grains are formed within anthers of the same flower; hence this character cannot be considered of any taxonomic value. Double fertilization occurs between 24 and 72 hr. after pollination. A cellular endosperm is formed, the peripheral layer acting as an absorbing tissue during the early ontogeny of the seed. Later this layer becomes organized as an aleurone layer and thereafter the source of nutrients is via the basal portion of the endosperm immediately adjacent to the apical end of the vascular tissue of the developing seed. Embryo development follows the Nicotiana variation of the Solanad type. The mature testa is composed of an outer layer of thick-walled epidermal cells, an inner layer of thin-walled cells and an intervening mass of disorganized tissue. In those instances where the ovule or young seed aborts, the endothelial cells of the integument become hyperactive and proliferate to such an extent that the space formerly occupied by the gametophyte or the developing endosperm and embryo becomes completely filled with endothelial tissue.     

A NOVEL PATTERN OF APICAL CELL POLYPLOIDY,SEQUENTIAL POLYPLOIDY REDUCTION AND INTERCELLULAR NUCLEAR TRANSFER IN THE RED ALGA POLYSIPHONIA

Nearly a century ago, Rosenvinge published a now-classic paper reporting nuclear transfer between cells of Polysiphonia during secondary pit connection (SPC) formation. While reinvestigating this phenomenon, we discovered that the uninucleate apical cell, which is the progenitor of all cells in the plant, has many times (ca. 64–128 ×) the level of nuclear DNA characteristic of nuclei of gametes or mature pericentral cells. Via a regular sequence of cell divisions, the polyploid apical cell gives rise to tiers of cells, each composed of a number of pericentral cells which surround a single central cell. A large proportion of the nuclear divisions are not accompanied by DNA replication. Thus, as the number of nuclei within elongating pericentral cells increases, the DNA level of nuclei in these cells “cascades” down to the DNA level expected for the particular life history generation (i.e., gametophyte or tetrasporophyte). In mature pericentral cells, the number of nuclei is proportional to the volume of the cell. The pattern of nuclear division, reduction in ploidy level and the timing of intercellular nuclear transfer via SPC formation is regular and characteristic of a species. Nuclei transferred from one cell to an adjacent cell participate in the further nuclear divisions of the recipient cell. The degree of polyploidy in apical cells may determine the number of cells in a “determinant” branch or even the number of cells in “indeterminant” axes. In addition, the highly polyploid state of the germinating spore and its pattern of development may provide for the rapid initial growth so characteristic of this taxon.     

Endoreduplication in cucumber (Cucumis sativus) seeds during development, after processing and storage, and during germination

Flow cytometry was used to study endoreduplication in developing, stored and germinating seeds of cucumber ( Cucumis sativus ). Fruits growing in a commercial seed production field were collected every 7 days, starting 14 days after pollination (DAP) up to 63 DAP (commercial harvest time). Seeds were isolated and the proportion of nuclei with different DNA contents in the whole seeds and in the embryos was analysed. Germination capacity of fresh and dried seeds at 25°C was established. In addition, the same analyses were performed on the seeds after processing (fermentation, drying and cleaning), following 1 and 2 years of storage, and after imbibition for 3, 6 and 12 h. In the young developing seeds, endoreduplication up to 128C occurred but this decreased to 8C by maturity. The proportion of endosperm nuclei was the highest at 21 DAP (30%) and then decreased to below 14% at harvest and 8% after processing. In the mature processed seeds, the majority of embryo nuclei (about 80%) contained 2C DNA; however, about 2% of endoreduplicated (8C) nuclei were still present. Seeds did not show any germination capacity up to 21 DAP; then it gradually increased to reach 100% as early as 49 DAP, 2 weeks before commercial harvest time. The relationship between seed maturity, germination and cell cycle status is discussed. The mean C-value of the seed cells as well as the (4C + 8C + 16C)/2C ratio are recommended as markers of cucumber seed maturity and the advancement of germination/priming (the stage of germination sensu stricto ).     

小麦受精过程的细胞化学研究

小麦成熟胚囊卵细胞中存在较多围核分布的淀粉粒和少量散布的脂类颗粒;两个助细胞中积累很多脂类,未见有淀粉粒存在;中央细胞中存在中等量均匀分布的淀粉粒和脂类颗粒。受精时期,胚囊内各细胞中淀粉粒变化不大。精卵核融合时,卵细胞和中央细胞中的脂类分别存在一个积累高峰。合子与相应时期游离核胚乳中的脂类颗粒均较少。原胚初期,每个原胚细胞及胚乳原生质中均积累较多脂类。珠孔附近的内珠被细胞中脂类颗粒较多,并存在一个有规律的变化。在观察的所有发育时期的胚珠中,均未发现贮存蛋白质。胚珠中脂类的一系列变化可能与雌性细胞的营养、胚胎发育初期的养料及花粉管的定向生长等有关     

Occurrence of Mitochondria in the Nuclei of Tobacco Sperm Cells

Tobacco sperm cells contain intact mitochondria within their nuclei with a frequency of 0.35 [plusmn] 0.13 per cell. These inclusions appear to originate from mitochondria found among chromatids in the highly elongated metaphase plate of the dividing generative cell. These organelles are apparently captured during the reconstitution of the nuclear envelope. Only sperm cells were observed to contain these nuclear mitochondria; generative cells, vegetative pollen cells, transmitting tissue cells, unfertilized egg cells, and central cells lacked them. Nuclear mitochondria were also seen in the nuclei of the egg and central cell after fusion with sperm nuclei, suggesting that nuclear mitochondria are transmitted into the zygote and primary endosperm cells during double fertilization. Organellar inclusions in the sperm nucleus provide a potential mechanism for transmitting organellar DNA into the next generation and could potentially facilitate the transfer of genetic material between the nucleus and other organelles.     

Changes in Nuclear DNA Content of Endosperm Cells During Grain Development in Rice (Oryza sativa)

Morphological, cytological and quantitative DNA changes associatedwith endosperm development in rice caryopsis were investigated.Following a brief free-nuclear phase, the endosperm became cellularby the 4th d after anthesis. While the mean length and breadthof grain attained maximum values at about 10, d after anthesis,f. wt of the whole grain, and of the endosperm separately, continuedto increase until about 16 d after anthesis. Cell divisionsin the endosperm continued until 10 d and following stabilizationof the cell number, the nuclei attained irregular shapes. Thesize of the nuclei and nuclei and the amount of nuclear DNAvaried considerably during endosperm development. The endospermnuclei did not retain the expected 3C–6C DNA level afterthe first few rounds of division and nuclei having more than30C DNA were frequent 8 d past anthesis. The highest C valuerecorded was 74C in a 16-d-old endosperm cell. Oryza sativa, rice, caryopsis, endosperm, cell number, nuclear area, nuclear DNA content, endoreduplication     

Multinucleate storage cells in Douglas-fir (Pseudotsuga menziesii (Mirbel) Franco) and the effect of seed parasitism by the chalcid Megastigmus spermotrophus Wachtl

Megagametophytes of Douglas-fir (Pseudotsuga menziesii (Mirbel) Franco) accumulated storage products following fertilization. As megagametophytes matured, the number of nuclei per cell rose, resulting in syncytial storage cells. Studies carried out on trees in France and Canada confirmed that such previously unreported, free nuclear cells were a normal part of late megagametophyte development. Unfertilized megagametophytes showed that some binucleate cells before degeneration resulted in empty seed. Insect parasitism prevented megagametophyte abortion in unfertilized ovules. Oviposition by a torymid chalcid wasp (Megastigmus spermotrophus Wachtl) early in megagametophyte development resulted in normal megagametophyte development. Around the time of plant egg maturation, binucleate and trinucleate cells were observed. As megagametophytes matured, multinucleate mature storage cells rich in proteins, lipids and starch were formed. The insect was able to induce identical nuclear behaviour in infested, unfertilized megagametophytes, as that of uninfested, fertilized megagametophytes.