Endopolyploidy in seed plants

Two main attempts have been suggested for the biological significance of endopolyploidy: (i) provision of high DNA amounts to support high synthetic demands in certain cells and (ii) compensation for a lack of nuclear DNA in species with small genomes. However, in seed plants, the positive correlation between DNA content and cell volume of endopolyploid cells suggests other possibilities. Cell size paralleled by the endopolyploidy level has an impact on growth and development. Endopolyploidy levels in turn are characteristic for a given species and even families, reflecting the adaptation to certain habitats during phylogeny. Furthermore, endopolyploidy levels vary to some degree between individuals of one species in response to different environmental conditions. In addition, endopolyploidy differs between different tissues suggests that a certain cell size is advantageous for a given cell function. This article reviews these findings and discusses more conclusive possible functions of endopolyploidy.     

DNA, RNA, protein and heterochromatin changes during embryo development and germination of soybean (Glycine max L.)

DNA, RNA, protein and heterochromatin were measured cytophotometrically in developing soybean (Glycine max) seeds. The average 2C DNA content for the soybean genome was 2.64 pg. The amounts of nuclear DNA in embryo axes showed no significant change during embryo development, whereas the DNA content in cotyledon nuclei increased significantly from 3.58 pg to 5.49 pg. The number of endopolyploid nuclei increased from 26% to 48% and the DNA content from 4.45 to 5.49 pg after cessation of cell division. The changes in RNA and protein content during embryo development were in general similar to those in DNA content. This can be interpreted that increased DNA levels in soybean cotyledons generated during embryogeny increase the protein synthesizing capacity. During the first 15 days of germination, the number of endopolyploid nuclei in cotyledons declined from 46% to 4%, and this decline is interpreted as DNA degradation providing a ready source of nucleosides and phosphates during early embryo growth. A later decline, however, between 15 and 20 days after germination, was age related similar to leaf senescence, because the percentage of endopolyploid nuclei remained unchanged while the number of non-viable cells increased. In senescing cotyledons, 73% and 80% of RNA and protein but only 20% of DNA were lost, as compared to dormant cotyledons. The heterochromatin (condensed chromatin) measurements indicated that nuclei of metabolically inactive dormant and senescent cotyledon nuclei contained an average of 33% more heterochromatin than nuclei from the green cotyledons of seedlings.     

The Growth of the Nucleus in Dividing and Non-dividing Cells of the Pea Root

The growth of the nucleus and the cell in the pea root was followedthrough the mitotic cycle and subsequently in post-mitotic developmentby comparing cells and nuclei from the meristem, at differentstages of interphase, and cells and nuclei from two regionsof the enlarging zone of the root. Measurements of cell andnuclear volumes were made in sections of fixed roots. Measurementsof nuclear volume, DNA content, and dry mass were made on isolatednuclei. Growth in the mitotic cycle was characterized by a doublingof DNA and nuclear dry mass and a five-fold increase of nuclearvolume. Since cell volume doubled, a differential hydrationof cytoplasm and nucleus is inferred. Post-mitotic growth wascharacterized by a four-fold or greater increase in cell volume,with vacuolation and a continued increase of cytoplasmic constituents,but a cessation of nuclear growth except by uptake of water;the only increase in nuclear dry matter appeared to be in cellsbecoming endopolyploid. The concentration of dry matter in thenucleus fell as the nuclei enlarged in the mitotic cycle andin post-mitotic growth. The relationships between the measuredparameters are examined to see whether they might be indicativeof causal relationships.     

Modal DNA values of normal and malignant urothelial cells of the bladder in relation to nuclear size

In a study of the correlation between mean nuclear size and DNA content in urinary bladder carcinoma, the modal DNA values of cell suspensions from 125 biopsies, obtained from 86 patients with malignant or normal urinary bladder epithelium, were analyzed by flow cytometry (FCM). Light microscopic measurements of nuclear size were carried out on smears from the same material. The results were correlated to the histopathologic stage and grade. The mean nuclear volumes were significantly larger in diploid tumor cells than in cells of normal epithelium. Aneuploid tumors showed significantly larger nuclei than did diploid tumors. Although there was a significant correlation between increases in the nuclear volume and in the DNA content, there was some overlapping between various grades of malignancy: mean nuclear volumes in aneuploid grade 2 tumors did not differ from those in aneuploid grade 3 tumors. A combination of FCM and morphometry discriminated all but 16% of the tumors from the normal cases. It is concluded that FCM and morphometry are complementary and can be used for the objective characterization of urinary bladder carcinomas.     

A comparative study in twelve mammalian species of volume densities, volumes, and numerical densities of selected testis components, emphasizing those related to the Sertoli cell

Morphometric studies were performed on 12 mammalian species (degu, dog, guinea pig, hamster, human, monkey, mouse, opossum, rabbit, rat, stallion, and woodchuck) to determine volume density percentage (Vv%), volume (V), and numerical density (Nv) of seminiferous tubule components, especially those related to the Sertoli cell, and to make species comparisons. For most species, measurements were taken both from stages where elongate spermatids were deeply embedded within the Sertoli cell and from stages near sperm release where elongate spermatids were in shallow crypts within the Sertoli cell. Montages, prepared from electron micrographs, were used to determine Vv% of Sertoli cell components in seminiferous tubules. Excluding the tubular lumen, the Sertoli cell occupied from a high of 43.1% (woodchuck) to a low of 14.0% (mouse) of the tubular epithelium. There was a strong negative correlation (r = -0.83; P less than 0.005) of volume occupancy of Sertoli cells with sperm production. Nuclear volume, as determined by serial reconstruction using serial thick sections, ranged from a high of 848.4 microns 3 (opossum) to a low of 273.8 microns 3 (degu). There was no correlation (r = 0.02) of nuclear volume with volume occupancy (Vv%) in the tubule. Sertoli cell volume was determined by point-counting morphometry at the electron-microscope level as the product of the nuclear size and points determined over the entire cell divided by points over the nucleus. Sertoli cell V ranged from 2,035.3 microns 3 (degu) to 7,011.6 microns 3 (opossum) and was highly correlated (r = 0.85; P less than 0.001) with nuclear size. However, there was no significant correlation between the Sertoli cell size (V) and volume occupancy (Vv%; r = 0.13) or sperm production (r = -0.21). Stereological estimates of the numerical density (Nv) of Sertoli cells ranged from a high of 101.9 x 10(6) (monkey) to a low of 24.9 x 10(6) (rabbit) cells per cm3 of testicular tissue. There was no correlation of numerical density of Sertoli cells with sperm production (r = 0.002). A negative correlation was, however, observed between the numerical density of the Sertoli cells and the Sertoli cell size (r = -0.79; P less than 0.002). Data from the present study are compared with those previously published. This is the first study to compare Sertoli cell morphological measurements using unbiased sampling techniques. Morphometric data are provided which will serve as a basis for other morphometric studies.     

Poly(ADP-ribose) polymerase activity in intact or permeabilized leukocytes from mammalian species of different longevity

Poly(ADP-ribosyl)ation is a eukaryotic posttranslational protein modification catalyzed by poly(ADP-ribose) polymerase (PARP), a highly conserved nuclear enzyme which uses NAD as substrate. We have previously tested PARP activity in permeabilized mononuclear blood cells (MNC) from 13 mammalian species as a function of the species-specific life span. A direct and maximal stimulus of PARP activation was provided by including saturating amounts of a double-stranded ollgonucleotide in the PARP-reaction buffer. The data yielded a strong positive correlation between PARP activities and the species' maximal life spans (r=0.84; p0.001). Here, we investigated the formation of poly(ADP-ribose) inliving MNC from two mammalian species with widely differing longevity (rat and man) by immunofluorescence detection of poly(ADP-ribose). The fraction of positive cells was recorded, following -irradiation of intact MNC, as a semiquantitative estimation of poly(ADP-ribose) formation. Human samples displayed a significantly higher percentage of positivity than did those from rats, consistent with our previous results on permeabilized cells. While rat MNC had a higher NAD content than human MNC, the number of radiation-induced DNA strand breaks was not significantly different in the two species. Since poly(ADP-ribosyl)ation is apparently involved in DNA repair and the cellular recovery from DNA damage, we speculate that the higher poly(ADP-ribosyl)ation capacity of long-lived species might more efficiently help to slow down the accumulation of unrepaired DNA damage and of genetic alterations, as compared with short-lived species. (Mol Cell Biochem138: 85–90, 1994)     

Chromosome studies in Hippeastrum (Amaryllidaceae): variation in genome size

This paper presents the karyotype and DNA content of 12 diploid species of Hippeastrum from South America. The variation in genome size is compared with the karyotype and DNA content of Amaryllis belladonna from South Africa. The Hippeastrum species present a uniform and bimodal basic karyotype formula, but significant differences are found in the total chromosome volume (TCV) and nuclear DNA content. A positive correlation between the DNA content and TCV is also observed. The karyotype's constancy is a product of changes in DNA content occurring in the whole chromosome complement. The DNA addition to the long and short sets of chromosomes varies independently. In species with higher DNA contents, the short chromosomes add equal DNA amounts to both arms, maintaining their metacentric morphology, whereas the long chromosomes add DNA only to the short arm, increasing the chromosome symmetry. These data show that the evolutionary changes in DNA amount are proportional to chromosome length, maintaining the karyotypic uniformity. A. belladonna has a larger DNA content and possesses a karyotype different from that of Hippeastrum spp., supporting the distinction between the two genera and upholding the name Amaryllis for the South African entity against Hippeastrum for the South American genus.  © 2007 The Linnean Society of London, Botanical Journal of the Linnean Society , 2007, 155 , 171–178.     

Loading of Arabidopsis centromeric histone CENH3 occurs mainly during G2 and requires the presence of the histone fold domain

The centromeric histone H3 (CENH3) substitutes histone H3 within the nucleosomes of active centromeres in all eukaryotes. CENH3 deposition at centromeres is needed to assemble the kinetochore, a complex of conserved proteins responsible for correct chromosome segregation during nuclear division. Histones of regular nucleosomes are loaded during replication in S phase, while CENH3 deposition deviates from this pattern in yeast, human, and Drosophila melanogaster cells. Little is known about when and how CENH3 targets centromeric loci. Therefore, we determined the location and quantity of recombinant enhanced yellow fluorescent protein (EYFP)-CENH3 in mitotic root and endopolyploid leaf nuclei of transgenic Arabidopsis thaliana cells. Our data indicate significant loading of A. thaliana CENH3 during G2 (before splitting into sister kinetochores) rather than during the S or M phase of the cell cycle. The histone fold domain of the C-terminal part of CENH3 is sufficient to target A. thaliana centromeres. A. thaliana EYFP-CENH3 can recognize and target three different centromeric repeats of Arabidopsis lyrata but not field bean (Vicia faba) centromeres.     

Genome size of man and animals relative to the plant Allium cepa

A direct Feulgen-cytophotometric comparison of the genomic DNA content (C value) was performed between the liliaceous plant species Allium cepa and a number of animal species to reassess the genome size ratios between plants and animals. These appeared unduly ambiguous as a consequence of divergent picogram estimates in several animal reference species. Taking 1C = 16.75 pg for Allium cepa, the estimates were (1C value in picograms): man, 3.11; Indian muntjak CCL 157 cell line, 2.68; domestic pig, 2.79; Chinese hamster, 2.66; CHO cell line, 2.73; laboratory rat, 2.65; mouse, 3.04; rat kangaroo Pt-K2 cell line, 4.21; fowl, 1.16; and the green toad, 4.30. These values are consistent with a number of independent absolute and relative DNA content determinations reported for animals, and therefore define a coherent set of animal and plant reference values for genome size determinations.     

Eukaryotic non-coding DNA is functional: evidence from the differential scaling of cryptomonad genomes

Genic DNA functions are commonplace: coding for proteins and specifying non-messenger RNA structure. Yet most DNA in the biosphere is non-genic, existing in nuclei as non-coding or secondary DNA. Why so much secondary DNA exists and why its amount per genome varies over orders of magnitude (correlating positively with cell volume) are central biological problems. A novel perspective on secondary DNA function comes from natural eukaryote eukaryote chimaeras (cryptomonads and chlorarachneans) where two phylogenetically distinct nuclei have coevolved within one cell for hundreds of millions of years. By comparing cryptomonad species differing 13-fold in cell volume, we show that nuclear and nucleomorph genome sizes obey fundamentally different scaling laws. Following a more than 125-fold reduction in DNA content, nucleomorph genomes exhibit little variation in size. Furthermore, the present lack of significant amounts of nucleomorph secondary DNA confirms that selection can readily eliminate functionless nuclear DNA, refuting 'selfish' and 'junk' theories of secondary DNA. Cryptomonad nuclear DNA content varied 12-fold: as in other eukaryotes, larger cells have extra DNA, which is almost certainly secondary DNA positively selected for a volume-related function. The skeletal DNA theory explains why nuclear genome size increases with cell volume and, using new evidence on nucleomorph gene functions, why nucleomorph genomes do not.     

Increased nuclear DNA content in raphide crystal idioblasts during development in Vanilla planifolia L. (Orchidaceae)

Longitudinal files of raphide crystal idioblasts form within the cortical meristematic region of Vanilla planifolia aerial roots. Cell and nuclear enlargement occur gradually throughout idioblast development and nuclear diameter approximates idioblast maturity. Cytophotometric determination of nuclear DNA (Feulgen) contents, measured by the two-wavelength method, revealed that all cortical parenchyma cells are diploid (2C = 6.3 pg), whereas all crystal idioblast nuclei are endopolyploid. Idioblast nuclear DNA content ranged from 4C to 32C (106 pg) and averaged 5.9 times that of parenchyma telophase nuclei. Frequency distribution of individual DNA content measurements depicts multiple genomes (increasing with geometric periodicity) to the 8C level, followed by less strict DNA replication within the crystal idioblast genome. The largest nuclei had the highest DNA content. Endomitotic stages of preprophasic heterochromatic dispersion (Z phase) and partial prophasic chromosomal coiling are observed with light and electron microscopy. DNA content values above the 8C level do not fit the geometrical order which is found if the total genome is replicated during each endo-cycle, a result indicating differential DNA replication. Chromocenter counts substantiate the occurrence of endomitosis to the 8C level and suggest heterochromatin underreplication in higher endopolyploid idioblast nuclei. Possible relationships between observed cytological events of idioblast development and nuclear condition are discussed.     

Characterization, expression and subcellular distribution of a novel MFP1 (matrix attachment region-binding filament-like protein 1) in onion

MFP1 (matrix attachment region-binding filament-like protein 1) is a conserved nuclear and chloroplast DNA-binding protein encoded by a nuclear gene, well characterized in dicot species. In monocots, only a 90 kDa MFP1-related protein had been characterized in the nucleus and nuclear matrix of Allium cepa proliferating cells. We report here a novel MFP1-related nuclear protein of 80 kDa in A. cepa roots, with M(r) and pI values similar to those of MFP1 proteins in dicot species, and which also displays a dual location, in the nucleus and chloroplasts of leaf cells. However, this novel protein is not a nuclear matrix component. It shows a spotted intranuclear distribution in small foci differing from the nuclear bodies containing the 90 kDa protein. In electron microscopy analysis, the intranuclear foci containing the 80 kDa MFP1 appeared as small loose structures at the periphery of condensed chromatin patches. This protein was also located in the nucleolus. It was abundant in meristematic cells, but its level fell when proliferation stopped. This different expression and distribution, and its preferential location at the boundaries between heterochromatin and euchromatin, suggest that the novel 80 kDa protein might be associated with decondensed DNA and could play a role in chromatin organization.     

Natural variation in nuclear characters of meristems in Vicia faba

The natural variation in chromosome size in root-tip and shoot apex meristems of Vicia faba has been studied. Chromosomes in main root-tips of one week old plants are 2–3 times larger than those in small lateral root-tips of 3 week old plants. While the nuclear DNA content remains constant, the nuclear RNA and nuclear histone contents show a positive linear correlation with chromosome volume. The DNA: histone ratio varies and is lowest in cells with large chromosomes. Nuclear volume and chromosome volume are not linearly related and changes in nuclear density therefore seem likely. A positive linear relationship between chromosome volume and mitotic index in colchicined squashes is demonstrated. The results strongly suggest that variation in chromosome size indicates a corresponding change in the rate of cell metabolism and may well reflect change in genetic activity.     

Nucleotype and cell size in vertebrates: a review

The relationships between genome size and various cell morphometric parameters have been assayed in 357 species of Vertebrates, in order to verify the existence and significance of the so-called "nucleotypic effect" in this subphylum. The results obtained clearly manifest a significant relationship between the increase in genome size and that in nuclear volume, nuclear surface, cell volume and cell surface. A precise correlation is also observed between the increase in DNA content and the decrease in the surface/volume ratios of the nucleus and the cell. Other parameters, such as the nucleoplasmic index and DNA concentration, though showing a slight increase with increasing genome size, have values rather homogeneous in each Vertebrate group. These results have allowed some interesting speculations on various problems; for example, the mechanisms through which genome size can influence the cell size; the influence of the DNA content and cell morphometric parameters on functional level of the cell and the organism; the importance of the nucleotypic effect in the adaptation to the environment of the various Vertebrate groups. From this study it seems possible to make the following conclusions: 1) in Vertebrates, genome size would exert a real nucleotypic influence on cell size; 2) genome sizes and cell morphometric parameters seem to be involved in the regulation of cell metabolism; 3) the regulation of some morphometric parameters depends strictly and automatically on the DNA amount or on other morphometric parameters. The regulation of others, instead, depends on the interaction of different factors, which do not always act synergically; 4) the nucleotypic effect seems to have different distribution and importance in Anamniotes and Amniotes.     

Significant correlations between atmospheric spectra according to Baumer and the in vitro incorporation of [3H]thymidine into the nuclear DNA of C6-glioma cells

Significant correlations between certain spectra of atmospherics (spherics) according to Baumer (a.t.B), i.e. naturally occurring electro-magnetic impulses in the range of 4-50 kHz, and several diseases or biological parameters have been published earlier. Now we show that there exists a highly significant negative correlation (r = -0.61, P greater than 0.004) between the occurrence of 28 kHz impulses (a.t.B.) and the in vitro incorporation of thymidine into the nuclear DNA of C6-glioma cells. The positive correlation with the 10 kHz impulses (a.t.B) (r = 0.39), however, is statistically not significant (P greater than 0.055).     

POT1-independent single-strand telomeric DNA binding activities in Brassicaceae

Telomeres define the ends of linear eukaryotic chromosomes and are required for genome maintenance and continued cell proliferation. The extreme ends of telomeres terminate in a single-strand protrusion, termed the G-overhang, which, in vertebrates and fission yeast, is bound by evolutionarily conserved members of the POT1 (protection of telomeres) protein family. Unlike most other model organisms, the flowering plant Arabidopsis thaliana encodes two divergent POT1-like proteins. Here we show that the single-strand telomeric DNA binding activity present in A. thaliana nuclear extracts is not dependent on POT1a or POT1b proteins. Furthermore, in contrast to POT1 proteins from yeast and vertebrates, recombinant POT1a and POT1b proteins from A. thaliana , and from two additional Brassicaceae species, Arabidopsis lyrata and Brassica oleracea (cauliflower), fail to bind single-strand telomeric DNA in vitro under the conditions tested. Finally, although we detected four single-strand telomeric DNA binding activities in nuclear extracts from B. oleracea , partial purification and DNA cross-linking analysis of these complexes identified proteins that are smaller than the predicted sizes of BoPOT1a or BoPOT1b. Taken together, these data suggest that POT1 proteins are not the major single-strand telomeric DNA binding activities in A. thaliana and its close relatives, underscoring the remarkable functional divergence of POT1 proteins from plants and other eukaryotes.     

DNA Content Variation and Its Significance in the Evolution of the Genus Micrasterias (Desmidiales,Streptophyta)

It is now clear that whole genome duplications have occurred in all eukaryotic evolutionary lineages, and that the vast majority of flowering plants have experienced polyploidisation in their evolutionary history. However, study of genome size variation in microalgae lags behind that of higher plants and seaweeds. In this study, we have addressed the question whether microalgal phylogeny is associated with DNA content variation in order to evaluate the evolutionary significance of polyploidy in the model genus Micrasterias. We applied flow-cytometric techniques of DNA quantification to microalgae and mapped the estimated DNA content along the phylogenetic tree. Correlations between DNA content and cell morphometric parameters were also tested using geometric morphometrics. In total, DNA content was successfully determined for 34 strains of the genus Micrasterias. The estimated absolute 2C nuclear DNA amount ranged from 2.1 to 64.7 pg; intraspecific variation being 17.4–30.7 pg in M. truncata and 32.0–64.7 pg in M. rotata. There were significant differences between DNA contents of related species. We found strong correlation between the absolute nuclear DNA content and chromosome numbers and significant positive correlation between the DNA content and both cell size and number of terminal lobes. Moreover, the results showed the importance of cell/life cycle studies for interpretation of DNA content measurements in microalgae.     

The skeletal function of non‐genic nuclear DNA: new evidence from ancient cell chimaeras

DNA can be divided functionally into three categories: (1) genes — which code for proteins or specify non-messenger RNAs; (2) semons — short specific sequences involved in the replication, segregation, recombination or specific attachments of chromosomes, or chromosome regions (e.g. loops or domains) or selfish genetic elements; (3) secondary DNA — which does not function by means of specific sequences. Probably more than 90% of DNA in the biosphere is secondary DNA present in the nuclei of plants and phytoplankton. The amount of genic DNA is related to the complexity of the organism, whereas the amount of secondary DNA increases proportionally with cell volume, and not with complexity. This correlation is most simply explained by the skeletal DNA hypothesis, according to which nuclear DNA functions as the basic framework for the assembly of the nucleus and the total genomic DNA content functions (together with relatively invariant folding rules) in determining nuclear volumes. Balanced growth during the cell cycle requires the cytonuclear ratio to be basically constant, irrespective of cell volume; thus nuclear volumes, and therefore the overall genome size, have to be evolutionarily adjusted to changing cell volumes for optimal function. Bacteria, mitochondria, chloroplasts and viruses have no nuclear envelope; and the skeletal DNA hypothesis simply explains why secondary DNA is essentially absent from them but present in large cell nuclei. Hitherto it has been difficult to refute the alternative hypothesis that nuclear secondary DNA (whether junk or selfish DNA) accumulates merely by mutation pressure, and that selection for economy is not strong enough to eliminate it, whereas accumulation in mitochondria and plastids is prevented by intracellular replicative competition between their multiple genomes. New data that discriminate clearly between these explanations for secondary DNA come from cryptomonads and chlorarachneans, two groups of algae that originated independently by secondary symbiogenesis (i.e., the merger of two radically different eukaryote cells) several hundred million years ago. In both groups the nucleus and plasma membrane of the former algal symbiont persist as the nucleomorphs and periplastid membrane, respectively. The fact that nucleomorphs have undergone a 200- to 1000-fold reduction in genome size and have virtually no secondary DNA shows that selection against non-functional nuclear DNA is strong enough to eliminate it very efficiently; therefore, the large amounts of secondary DNA in the former host nuclei of these chimaeras, and in nuclei generally, must be being maintained by positive selection. The divergent selection for secondary DNA in the nucleus and against it in nucleomorphs is readily explicable by the skeletal DNA hypothesis, given the different spectrum of gene functions that it encodes.This revised version was published online in October 2005 with corrections to the Cover Date.