Multinucleate plant cells : II. Requirements for nuclear DNA synthesis

A synchronous population of multinucleate cells has been experimentally induced in Allium cepa root meristems by a chemical method that involves the induction of aneuploidy in individual nuclei. Whereas all multinucleate cells as a whole present a tetraploid chromosome complement, their aneuploid nuclei constitute compartments containing only part of the parental genetic material. We analysed the degree of synchrony in the course of S period progression in the unbalanced nuclei sharing a common cytoplasm (bi-, tri- and tetranucleate cells). In most of the multinucleate cells the nuclei entered DNA replication simultaneously, but a significant proportion of cells showed labelled and unlabelled nuclei which was due to the absence of DNA synthesis in some aneuploid nuclei. These nuclei lacked replicative capacity, but did not inhibit DNA synthesis of the replicating nuclei. Our findings confirm the existence of cytoplasmic factor(s) inducing the synchronous initiation of S period, and suggest strongly the occurrence of intranuclear factor(s)—likely gene products—as a requirement for the onset of DNA synthesis itself in every nucleus.     

Patterns of DNA and chromosome variation during in vitro growth in various genotypes of potato

Patterns of variation in nuclear DNA content and chromosome number were analysed in a temporal sequence, during in vitro growth of calli and cell suspensions in two monohaploids, a dihaploid and a tetraploid of potato (Solanum tuberosum). The results showed that both polyploidization and aneuploidy occurred during the initial stages of callus induction in all the genotypes. With further growth of callus, the frequency and extent of polyploidy and aneuploidy increased. In addition, the patterns of DNA and chromosome variation in cell suspension cultures revealed continued mitotic activity and transmission of cells with higher ploidy levels and aneuploidy. The results suggest that endoreduplication as well as endomitosis are important mechanisms of polyploidization, and that chromosome lagging and non-disjunction contribute to the production of aneuploidy.The various genotypes cultured under the same in vitro growth conditions differed in genetic instability, as assessed from the rate and degree of polyploidization and aneuploidy. Monohaploids showed more rapid rate of polyploidization than the dihaploid and tetraploid potatoes. It was concluded that the differences in genetic stability were due to different ploidy levels and genetic make-up of the genotypes.     

Syncytia in plants: cell fusion in endosperm—placental syncytium formation in Utricularia (Lentibulariaceae)

The syncytium formed by Utricularia is extremely unusual and perhaps unique among angiosperm syncytia. All typical plant syncytia (articulated laticifers, amoeboid tapetum, the nucellar plasmodium of river weeds) are formed only by fusion of sporophytic cells which possess the same genetic material, unlike Utricularia in which the syncytium possesses nuclei from two different sources: cells of maternal sporophytic nutritive tissue and endosperm haustorium (both maternal and paternal genetic material). How is this kind of syncytium formed and organized and is it similar to other plant syncytial structures? We used light and electron microscopy to reconstruct the step-by-step development of the Utricularia syncytia. The syncytia of Utricularia developed through heterotypic cell fusion involving the digestion of the cell wall, and finally, heterokaryotic multinucleate structures were formed, which possessed different-sized nuclei that were not regularly arranged in the cytoplasm. We showed that these syncytia were characterized by hypertrophy of nuclei, abundant endoplasmic reticulum and organelles, and the occurrence of wall ingrowths. All these characters testify to high activity and may confirm the nutritive and transport functions of the syncytium for the developing embryo. In Utricularia, the formation of the syncytium provides an economical way to redistribute cell components and release nutrients from the digested cell walls, which can now be used for the embryo, and finally to create a large surface for the exchange of nutrients between the placenta and endosperm.     

Genome ploidy in different stages of the Giardia lamblia life cycle

The early diverging eukaryotic parasite Giardia lamblia is unusual in that it contains two apparently identical nuclei in the vegetative trophozoite stage. We have determined the nuclear and cellular genome ploidy of G. lamblia cells during all stages of the life cycle. During vegetative growth, the nuclei cycle between a diploid (2N) and tetraploid (4N) genome content and the cell, consequently, cycles between 4N and 8N. Stationary phase trophozoites arrest in the G₂ phase with a ploidy of 8N (two nuclei, each with a 4N ploidy). On its way to cyst formation, a G₁ trophozoite goes through two successive rounds of chromosome replication without an intervening cell division event. Fully differentiated cysts contain four nuclei, each with a ploidy of 4N, resulting in a cyst ploidy of 16N. The newly excysted cell, for which we suggest the term 'excyzoite', contains four nuclei (cellular ploidy 16N). In a reversal of the events occurring during encystation, the excyzoite divides twice to form four trophozoites containing two diploid nuclei each. The formation of multiple cells from a single cyst is likely to be one of the main reasons for the low infectious doses of G. lamblia .     

Proliferation of multinucleate cells with unbalanced nuclei

When onion root meristems are treated with gamma-hexachlorocyclohexane the anaphase chromatids are distributed in discrete unbalanced groups and subsequent inhibition of cytokinesis in these cells produced a synchronous population of viable multinucleate cells with two, three of four aneuploid nuclei. When we compare the duration of G1, S and G2 periods in diploid cells with that obtained for multinucleate cells in the present study it seems clear that the differences, if they occur, are negligible. These results are consistent with the hypothesis that the cell mass/genome ratio can play an essential role in controlling cycle rate and that most of the genic requirements for interphase development must complement between the nuclei sharing a common cytoplasm, even though some factor inside every nucleus appears to be required for replicative capacity to be effective.     

Vertical heterogeneity of DNA ploidy level assessed by flow cytometry in calli of Passiflora Cincinnata

During in vitro culture conditions, callus tissue is exposed to different intensities of environmental stress, which may induce somaclonal variation. Among the possible resulting abnormalities, callus cells can exhibit distinct DNA ploidy levels, a type of somaclonal variation associated with euploidy and/or aneuploidy. As somaclonal variation has been regarded as both a positive and negative phenomenon, the development of strategies to carefully assess the stability of DNA ploidy level within callus tissue is highly valuable. To this end, the present work aimed to evaluate the presence of intra- and inter-calli heterogeneity in relation to DNA ploidy level and nuclei density by flow cytometry. Calli were induced from cotyledonary leaves of Passiflora cincinnata, a wild passion fruit species. Embryogenic friable calli cultivated for 2, 6, and 9 mo were classified as young, intermediary, and old, respectively. These calli were horizontally sliced from the bottom-up at approximately the same thickness, and a total of 160 layers were evaluated by flow cytometry. Inter- and intra-calli heterogeneities were detected in relation to nuclei density and DNA ploidy level. Additional analysis was performed to identify the most proliferative layer. We conclude that care must be taken when using callus as source material for flow cytometry, since one portion cannot represent the whole cell mass. Moreover, in order to prevent the emergence of undesired ploidies during clonal propagation, callus culture time should not be prolonged.     

Organisation of the endosperm and endosperm–placenta syncytia in bladderworts (Utricularia, Lentibulariaceae) with emphasis on the microtubule arrangement

Multinucleate cells play an important role in higher plants, especially during reproduction; however, the configurations of their cytoskeletons, which are formed as a result of mitosis without cytokinesis, have mainly been studied in coenocytes. Previous authors have proposed that in spite of their developmental origin (cell fusion or mitosis without cytokinesis), in multinucleate plant cells, radiating microtubules determine the regular spacing of individual nuclei. However, with the exception of specific syncytia induced by parasitic nematodes, there is no information about the microtubular cytoskeleton in plant heterokaryotic syncytia, i.e. when the nuclei of fused cells come from different cell pools. In this paper, we describe the arrangement of microtubules in the endosperm and special endosperm–placenta syncytia in two Utricularia species. These syncytia arise from different progenitor cells, i.e. cells of the maternal sporophytic nutritive tissue and the micropylar endosperm haustorium (both maternal and paternal genetic material). The development of the endosperm in the two species studied was very similar. We describe microtubule configurations in the three functional endosperm domains: the micropylar syncytium, the endosperm proper and the chalazal haustorium. In contrast to plant syncytia that are induced by parasitic nematodes, the syncytia of Utricularia had an extensive microtubular network. Within each syncytium, two giant nuclei, coming from endosperm cells, were surrounded by a three-dimensional cage of microtubules, which formed a huge cytoplasmic domain. At the periphery of the syncytium, where new protoplasts of the nutritive cells join the syncytium, the microtubules formed a network which surrounded small nuclei from nutritive tissue cells and were also distributed through the cytoplasm. Thus, in the Utricularia syncytium, there were different sized cytoplasmic domains, whose architecture depended on the source and size of the nuclei. The endosperm proper was isolated from maternal (ovule) tissues by a cuticle layer, so the syncytium and chalazal haustorium were the only way for nutrients to be transported from the maternal tissue towards the developing embryo.     

Multinucleate plant cells: I. Aneuploidy in a proliferating population

A new method for the production of multinucleate plant cells in meristematic populations is described. This method involves the aneuploidy induction of nuclei of a same cell. Allium cepa root tips were chemically treated, and the multinucleate cells obtained were scored at interphase and mitosis. When meristematic cells are treated with γ-hexachlorocyclohexane in appropriate culture conditions, the anaphase chromatids are distributed into discrete unbalanced groups. This phenomenon has been profited for inducing viable multinucleate cells with aneuploid nuclei after cytokinesis inhibition with caffeine.     

CYTOLOGY OF HELMINTHOSPORIUM TURCICUM AND ITS ASCIGEROUS STAGE,TRICHOMETASPHAERIA TURCICA

Knox- Davies , P. S., and J. G. Dickson . (U. Wisconsin, Madison.) Cytology of Helmintho sporium turcicum and its ascigerous stage, Trichometasphaeria turcica . Amer. Jour. Bot. 47(5) : 328—339. Illus. 1960.–The cells of the vegetative hyphae were generally multinucleate. Interphase nuclei resembled those of higher organisms, with a matrix of thread-like chromatin material surrounding a spherical nucleolus. “Beaked” nuclei frequently associated with anastomosing hyphae were interpreted as migrating nuclei. Nuclear division in the vegetative hyphae was rapid. Various division stages were distinguished but it was difficult to make accurate chromosome counts. The nucleoli were discarded at prophase or prometaphase and were reorganized in daughter nuclei at telophase. An outstanding feature of nuclear division was that all the nuclei in a cell divided simultaneously. Conidiophores and conidia were occasionally joined by wide cytoplasmic connections. They were multinucleate throughout their development. Mechanisms therefore exist for the perpetuation of heterokaryons through the conidium. Ascus development was studied in a hybrid between a dark and an albino isolate. Crozier formation was typical and nuclear fusion occurred in the young ascus. Four nuclear divisions were completed in the ascus before there was evidence of ascospore delimitation. Further nuclear division took place in the ascospores whose cells were multinucleate. The occurrence of less than 8 ascospores in an ascus appeared to follow degeneration of nuclei rather than the incorporation of a number of division-Ill nuclei in a single ascopore. Chromosome counts and irregularities in the appearance and behavior of nuclei and chromosomes in the asci indicate that aneuploidy occurs in Trichometasphaeria turcica. It is suggested that aneuploidy is a common phenomenon in the conidial stage of the fungus H. turcicum, and possibly also in other imperfect fungi.     

Polyamine starvation causes parallel increase in nuclear and chromosomal aberrations in a polyamine-dependent strain of CHO

Deprivation of polyamines and ornithine causes in a polyamine-dependent CHO strain aneuploidy and alterations in nuclear morphology including micronuclei, macronuclei, framented and bulged nuclei. There is also formation of multinucleate cells. The number of micronuclei and certain other nuclear aberrations increase concomitantly with chromosome abberrations.     

Synchronous nuclear-envelope breakdown and anaphase onset in plant multinucleate cells

Summary Multinucleate plant cells with genetically balanced nuclei can be generated by inhibiting cytokinesis in sequential telophases. These cells can be used to relate the effect of changes in the distribution of nuclei in the cytoplasm to the control of the timing of cell cycle transitions. Which mitotic cell cycle events are sensitive to differences in the, amount of cytoplasm surrounding each chromosomal complement has not been determined. To address this, we maximized the cell size by transiently inhibiting replication, while cell growth was not affected. The nuclei of 93% of the elongated cells reached prophase asynchronously compared to 46% of normal-sized multinucleate cells. The asynchronous prophases of normal-sized cells became synchronous at the time of nuclear-envelope breakdown, and the ensuing metaphase plate formation and anaphase onset and progression occurred synchronously. The elongated multinucleate cells were also very efficient in synchronizing the prophases at nuclear-envelope breakdown, in the prophase-to-prometaphase transition. However, 2.4% of these cells broke down the nuclear envelope asynchronously, though they became synchronous at the metaphase-to-anaphase transition. The kinetochore-microtubular cycle, responsible for coordinating the metaphase-to-anaphase transition and for the rate of sister segregation to opposite spindle poles during anaphase, remained strictly controlled and synchronous in the different mitoses of a single cell, independently of differences in the amount of cytoplasm surrounding each mitosis or its ploidy. Moreover, the degree of chromosome condensation varied considerably within the different mitotic spindles, being higher in the mitoses with the largest surrounding cytoplasm.Abbreviation NEB nuclear-envelope breakdown     

Karyotypic determinants of chromosome instability in aneuploid budding yeast

Recent studies in cancer cells and budding yeast demonstrated that aneuploidy, the state of having abnormal chromosome numbers, correlates with elevated chromosome instability (CIN), i.e. the propensity of gaining and losing chromosomes at a high frequency. Here we have investigated ploidy- and chromosome-specific determinants underlying aneuploidy-induced CIN by observing karyotype dynamics in fully isogenic aneuploid yeast strains with ploidies between 1N and 2N obtained through a random meiotic process. The aneuploid strains exhibited various levels of whole-chromosome instability (i.e. chromosome gains and losses). CIN correlates with cellular ploidy in an unexpected way: cells with a chromosomal content close to the haploid state are significantly more stable than cells displaying an apparent ploidy between 1.5 and 2N. We propose that the capacity for accurate chromosome segregation by the mitotic system does not scale continuously with an increasing number of chromosomes, but may occur via discrete steps each time a full set of chromosomes is added to the genome. On top of such general ploidy-related effect, CIN is also associated with the presence of specific aneuploid chromosomes as well as dosage imbalance between specific chromosome pairs. Our findings potentially help reconcile the divide between gene-centric versus genome-centric theories in cancer evolution.     

倍性复杂植物的2种倍性鉴定方法的建立及应用(专题约稿）

研究以猕猴桃属内不同植物的幼嫩叶片为材料,利用流式细胞术和全基因组SNP(single nucleotide polymorphism)位点杂合子等位基因深度比率(heterozygous allele depth ratio)分布2种方法进行猕猴桃倍性鉴定。对取样叶片的生长状态、防止细胞核黏连的PVP(聚乙烯吡咯烷酮)浓度、滤网目数及过滤次数、不同倍性样本全基因组SNP分型的参数调整等因素进行探索。结果表明,流式细胞术检测中取未展开的幼嫩叶片获得完整细胞核的数目最多;5%PVP对减少细胞核之间的黏连最适宜;500目滤网过滤3次效果最好。SNP的分型主要与模拟基因组的组装质量和过滤识别SNP的参数设置有关。流式细胞术鉴定倍性的关键技术是使用未展开的幼嫩叶片以保证足够数量的完整细胞核及减少细胞核之间的黏连。同一植物材料的染色体倍性在60Co-γ辐照处理前后未发生改变。全基因组SNP位点杂合子频率分布图判断的倍性与流式细胞术鉴定结果一致。2种鉴定结果可以相互验证,使倍性的判断变得更加准确,为加快猕猴桃育种提供了基础。     

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     

Genetic variation in aneuploidy prevalence and tolerance across Saccharomyces cerevisiae lineages

Individuals carrying an aberrant number of chromosomes can vary widely in their expression of aneuploidy phenotypes. A major unanswered question is the degree to which an individual’s genetic makeup influences its tolerance of karyotypic imbalance. Here we investigated within-species variation in aneuploidy prevalence and tolerance, using Saccharomyces cerevisiae as a model for eukaryotic biology. We analyzed genotypic and phenotypic variation recently published for over 1,000 S. cerevisiae strains spanning dozens of genetically defined clades and ecological associations. Our results show that the prevalence of chromosome gain and loss varies by clade and can be better explained by differences in genetic background than ecology. The relationships between lineages with high aneuploidy frequencies suggest that increased aneuploidy prevalence emerged multiple times in S. cerevisiae evolution. Separate from aneuploidy prevalence, analyzing growth phenotypes revealed that some genetic backgrounds—such as the European Wine lineage—show fitness costs in aneuploids compared to euploids, whereas other clades with high aneuploidy frequencies show little evidence of major deleterious effects. Our analysis confirms that chromosome gain can produce phenotypic benefits, which could influence evolutionary trajectories. These results have important implications for understanding genetic variation in aneuploidy prevalence in health, disease, and evolution.     

Endopolyploidy Changes with Age-Related Polyethism in the Honey Bee,Apis mellifera

Honey bees (Apis mellifera) exhibit age polyethism, whereby female workers assume increasingly complex colony tasks as they age. While changes in DNA methylation accompany age polyethism, other DNA modifications accompanying age polyethism are less known. Changes in endopolyploidy (DNA amplification in the absence of cell division) with increased larval age are typical in many insect cells and are essential in adults for creating larger cells, more copies of essential loci, or greater storage capacity in secretory cells. However, changes in endopolyploidy with increased adult worker age and polyethism are unstudied. In this study, we examined endopolyploidy in honey bee workers ranging in age from newly emerged up to 55 days old. We found a nonsignificant increase in ploidy levels with age (P < 0.1) in the most highly endopolyploid secretory cells, the Malpighian tubules. All other cell types decreased ploidy levels with age. Endopolyploidy decreased the least amount (nonsignificant) in neural (brain) cells and the stinger (P < 0.1). There was a significant reduction of endopolyploidy with age in leg (P < 0.05) and thoracic (P < 0.001) muscles. Ploidy in thoracic muscle dropped from an average of 0.5 rounds of replication in newly emerged workers to essentially no rounds of replication (0.125) in the oldest workers. Ploidy reduction in flight muscle cells is likely due to the production of G1 (2C) nuclei by amitotic division in the multinucleate striated flight muscles that are essential to foragers, the oldest workers. We suggest that ploidy is constrained by the shape, size and makeup of the multinucleate striated muscle cells. Furthermore, the presence of multiple 2C nuclei might be optimal for cell function, while higher ploidy levels might be a dead-end strategy of some aging adult tissues, likely used to increase cell size and storage capacity in secretory cells.     

Ploidy variation as an adaptive mechanism in human pathogenic fungi

Changes in ploidy have a profound and usually negative influence on cellular viability and proliferation, yet the vast majority of cancers and tumours exhibit an aneuploid karyotype. Whether this genomic plasticity is a cause or consequence of malignant transformation remains uncertain. Systemic fungal pathogens regularly develop aneuploidies in a similar manner during human infection, often far in excess of the natural rate of chromosome nondisjunction. As both processes fundamentally represent cells evolving under selective pressures, this suggests that changes in chromosome number may be a concerted mechanism to adapt to the hostile host environment. Here, we examine the mechanisms by which aneuploidy and polyploidy are generated in the fungal pathogens Candida albicans and Cryptococcus neoformans and investigate whether these represent an adaptive strategy under severe stress through the rapid generation of large-scale mutations. Insights into fungal ploidy changes, strategies for tolerating aneuploidies and proliferation during infection may yield novel targets for both antifungal and anticancer therapies.     

Do ploidy level and nuclear genome size and latitude of origin modify the expression of <Emphasis Type="Italic">Phragmites australis</Emphasis> traits and interactions with herbivores?

We studied the relationship between genome size and ploidy level variation and plant traits for the reed grass Phragmites australis. Using a common garden approach on a global collection of populations in Aarhus, Denmark, we investigated the influence of monoploid genome size and ploidy level on the expression of P. australis growth, nutrition and herbivore-defense traits and whether monoploid genome size and ploidy level play different roles in plant trait expression. We found that both monoploid genome size and latitude of origin contributed to variation in traits that we studied for P. australis, with latitude of origin being generally a better predictor of trait values and that ploidy level and its interaction with monoploid genome size and latitude of origin also contributed to trait variation. We also found that for four traits, tetraploids and octoploids had different relationships with the monoploid genome size. While for tetraploids stem height and leaf water content showed a positive relationship with monoploid genome size, octoploids had a negative relationship with monoploid genome size for stem height and no relationship for leaf water content. As genome size within octoploids increased, the number of aphids colonizing leaves decreased, whereas for tetraploids there was a quadratic, though non-significant, relationship. Generally we found that tetraploids were taller, chemically better defended, had a greater number of stems, higher leaf water content, and supported more aphids than octoploids. Our results suggest trade-offs among plant traits mediated by genome size and ploidy with respect to fitness and defense. We also found that the latitude of plant origin is a significant determinant of trait expression suggesting local adaptation. Global climate change may favor some genome size and ploidy variants that can tolerate stressful environments due to greater phenotypic plasticity and to fitness traits that vary with cytotype which may lead to changes in population genome sizes and/or ploidy structure, particularly at species’ range limits.     

Cytogeography and genome size variation in the Claytonia perfoliata (Portulacaceae) polyploid complex

Background and Aims

Genome duplication is a central process in plant evolution and contributes to patterns of variation in genome size within and among lineages. Studies that combine cytogeography with genome size measurements contribute to our basic knowledge of cytotype distributions and their associations with variation in genome size.

Methods

Ploidy and genome size were assessed with direct chromosome counts and flow cytometry for 78 populations within the Claytonia perfoliata complex, comprised of three diploid taxa with numerous polyploids that range to the decaploid level. The relationship between genome size and temperature and precipitation was investigated within and across cytotypes to test for associations between environmental factors and nuclear DNA content.

Key Results

A euploid series (n = 6) of diploids to octoploids was documented through chromosome counts, and decaploids were suggested by flow cytometry. Increased variation in genome size among populations was found at higher ploidy levels, potentially associated with differential contributions of diploid parental genomes, variation in rates of genomic loss or gain, or undetected hybridization. Several accessions were detected with atypical genome sizes, including a diploid population of C. parviflora ssp. grandiflora with an 18 % smaller genome than typical, and hexaploids of C. perfoliata and C. parviflora with genomes 30 % larger than typical. There was a slight but significant association of larger genome sizes with colder winter temperature across the C. perfoliata complex as a whole, and a strong association between lower winter temperatures and large genome size for tetraploid C. parviflora.

Conclusions

The C. perfoliata complex is characterized by polyploids ranging from tetraploid to decaploid, with large magnitude variation in genome size at higher ploidy levels, associated in part with environmental variation in temperature.