Endopolyploidy in seed plants is differently correlated to systematics, organ, life strategy and genome size

Endopolyploidy is a systemic feature in seed plants. A negative correlation between genome size and endopolyploidization has been claimed previously, assuming that a minimum amount of DNA, necessary for certain cell functions, has to be acquired by endopolyploidization of the corresponding cells in plants with small genomes. This assumption was based on the analysis of only a limited set of data from few species. In the present study the endopolyploidization of several organs of 54 seed plant species belonging to two gymnosperm and 14 angiosperm families was investigated. The results revealed a low negative correlation between genome size and endopolyploidization. However, differences between the families, between the different organs of a given species and between the different life‐cycle types with regard to endopolyploidization became obvious. A three‐way analysis of variance with covariate to quantify the impact of the different factors on the extent of endopolyploidization suggested that taxonomic position is the major factor determining the degree of endopolyploidy within a species, while life cycle, genome size and organ type have a minor but also significant effect on endopolyploidization. The comparison of habitats of 16 investigated Central European species implies that endopolyploidization represents a mean to accelerate the growth of plant species in niches, which require and support fast development.     

Endopolyploidy inHelianthus annuus (Asteraceae), A scanning cytophotometric study

Endopolyploidy has been detected in some varieties ofHelianthus annuus L. (Asteraceae/Compositae) by means of scanning photometry of Feulgen-stained nuclei and analysis of nuclear structure. In the hypocotyl cells of seedlings, ploidy levels reach respectively 8 C and 16 C in different varieties, in the root cells 8 C and 16 C; in the cotyledons of ripening seeds 4 C to 8 C values have been found, while all nuclei of the inflorescence axis of one variety exhibit a DNA content of 4 C.—This is the first report of endopolyploidy in a non-succulentAsteraceae species. The characteristic distribution of the endopolyploidy levels in different varieties suggests a strong genetic and/or hormonal control of the final nuclear DNA content in differentiated cells.     

Relationship between Endopolyploidy and Cell Size in Epidermal Tissue of Arabidopsis

Relative quantities of DNA in individual nuclei of stem and leaf epidermal cells of Arabidopsis were measured microspectrofluorometrically using epidermal peels. The relative ploidy level in each nucleus was assessed by comparison to root tip mitotic nuclei. A clear pattern of regular endopolyploidy is evident in epidermal cells. Guard cell nuclei contain levels of DNA comparable to dividing root cells, the 2C level (i.e., one unreplicated copy of the nuclear DNA). Leaf trichome nuclei had elevated ploidy levels of 4C, 8C, 16C, 32C, and 64C, and their cytology suggested that the polyploidy represents a form of polyteny. The nuclei of epidermal pavement cells were 2C, 4C, and 8C in stem epidermis, and 2C, 4C, 8C, and 16C in leaf epidermis. Morphometry of epidermal pavement cells revealed a direct proportionality between nuclear DNA level and cell size. A consideration of the development process suggests that the cells of highest ploidy level are developmentally oldest; consequently, the developmental pattern of epidermal tissues can be read from the ploidy pattern of the cells. This observation is relevant to theories of stomate spacing and offers opportunities for genetic analysis of the endopolyploidy/polyteny phenomenon.     

Endopolyploidy as a potential driver of animal ecology and evolution

Endopolyploidy – the existence of higher‐ploidy cells within organisms that are otherwise of a lower ploidy level (generally diploid) – was discovered decades ago, but remains poorly studied relative to other genomic phenomena, especially in animals. Our synthetic review suggests that endopolyploidy is more common in animals than often recognized and probably influences a number of fitness‐related and ecologically important traits. In particular, we argue that endopolyploidy is likely to play a central role in key traits such as gene expression, body and cell size, and growth rate, and in a variety of cell types, including those responsible for tissue regeneration, nutrient storage, and inducible anti‐predator defences. We also summarize evidence for intraspecific genetic variation in endopolyploid levels and make the case that the existence of this variation suggests that endopolyploid levels are likely to be heritable and thus a potential target for natural selection. We then discuss why, in light of evident benefits of endopolyploidy, animals remain primarily diploid. We conclude by highlighting key areas for future research such as comprehensive evaluation of the heritability of endopolyploidy and the adaptive scope of endopolyploid‐related traits, the extent to which endopolyploid induction incurs costs, and characterization of the relationships between environmental variability and endopolyploid levels.     

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.     

In planta quantification of endoreduplication using fluorescent in situ hybridization (FISH)

Endopolyploidy, i.e. amplification of the genome in the absence of mitosis, occurs in many plant species and happens along with organ and cell differentiation. Deciphering the functional roles of endopolyploidy is hampered by the fact that polyploid tissues generally comprise cells with various ploidy levels. In some fleshy fruits (amongst them tomato fruit) the ploidy levels present at the end of development range from 2C to 256C in the same tissue. To investigate the temporal and spatial distribution of endopolyploidy it is necessary to address the DNA content of individual nuclei in situ. Conventional methods such as fluorometry or densitometry can be used for some tissues displaying favorable characteristics, e.g. small cells, small nuclei, organization in a monolayer, but high levels of varying polyploidy are usually associated with large sizes of nuclei and cells, in a complex three dimensional (3-D) organization of the tissues. The conventional methods are inadequate for such tissue, becoming semi-quantitative and imprecise. We report here the development of a new method based on fluorescent in situ bacterial artificial chromosome hybridizations that allows the in situ determination of the DNA ploidy level of individual nuclei. This method relies on the counting of hybridization signals and not on intensity measurements and is expected to provide an alternative method for mapping endopolyploidy patterns in mature, 3-D organized plant tissues as illustrated by the analysis of ploidy level and cell size in pericarp from mature green tomato fruit.     

Endopolyploidy as a morphogenetic factor of development

This paper summarizes the works published by author and his co-workers in the Russian journal Tsitologiya concerning endopolyploidy in mollusks and appraises this phenomenon in general. Both ontogenetic and phylogenetic aspects of endopolyploidy have been studied. In the snail Succinea lauta, a complex examination of endomitosis has been performed. A regular replacement of the normal (complete) proliferative mitosis by abnormal (incomplete) restitutional mitosis, and then by Geitler's classic endomitosis has been demonstrated. We examined 29 bivalve and 82 gastropod species for the presence of polyploid cells in glandular tissues and ganglia. In the bivalve species, ordinary diploid cells form various tissues, while in the gastropods, the role of polyploidy in tissue development appears to increase in phylogenesis. The rise of endopolyploidy and cell giantism in histogeneses of a variety of animal and plant species is widely known. It is believed to be a regular event in the evolution of certain groups. To give a universal interpretation of endopolyploidy, we proposed that a single polyploid cell be better considered as an endoclone. In this case, evolutionary transformation of diploid cell clones into polyploid endoclones may be viewed as Dogel's oligomerization applied to cell-tissue level. From this viewpoint, major properties of an oligomerized system (intensification of function, functional efficiency (ergonomy), increased genomes reliability, simplification of the intra- and supersystem regulations, and acceleration of development) can be considered as principal peculiarities of polyploid growth strategy. The above peculiarities allow one to consider endopolyploidy as an additional means of integrative onto(histo)genetic regulations and correlations and as an important evolutionary factor (coordinations) acting through natural selection. Thus, in general, endopolyploidy is an adaptive morphogenetic factor, but its concrete role may differ in different tissues and organisms depending on cell specialization and histogenetic particularities.     

Evolutionary relationship of fat body endoreduplication and queen fecundity in termites

Endoreduplication or nuclear genome replication without cell division is widely observed in the metabolically active tissues of plants and animals. The fat body cells of adult female insects produce abundant yolk proteins and become polyploid, which is assumed to accelerate egg production. Recently, it was reported that in termites, endopolyploidy in the fat body occurs only in queens but not in the other females; however, the relationship between the fecundity and ploidy level in the fat body remains unclear. Termite queens exhibit a huge variation in their egg‐producing capacity among different species; queens in the species with a foraging lifestyle, in which workers leave the nest to forage outside, are much more fecund than those in the species living in a single piece of wood. In this study, we conducted ploidy analyses on three foragings and three wood‐dwelling termites via flow cytometry. In all the species, the fat body of queens contained significantly more polyploid cells than that of other nonreproductive females, considering their body size effect. However, the male fat body, which is not involved in yolk production, did not show consistency in polyploid cell numbers among the species studied. Moreover, highly fecund queens in foraging termites exhibit higher levels of endopolyploidy in their fat body than those with less fecundity in wood‐dwelling termites. These results suggest that endopolyploidy in the fat body of termite queens can boost their egg production, and the level of endopolyploidy in their fat body is linked to their fecundity. Our study provides a novel insight into the evolutionary relationship between endoreduplication and caste specialization in social insects.     

Endopolyploidie und Chloroplastenzahlen in verschiedenartigen Zellen trisomer Zuckerrüben

Summary Earlier investigations were continued on cell specific effects of eight different extra chromosomes in single trisomic sugar beets (Beta vulgaris L.). The degree of endopolyploidy, which was determined approximately by means of chloroplast numbers per cell, was changed by the presence of certain extra chromosomes: it either increased (I, II, VIII) or decreased (III through VII), but in epidermis and in spongy parenchyma the change was in the same direction. Sometimes, however, the degree was found to increase in spongy parenchyma alone or to decrease in epidermis alone; this evidence is consistent with the generally low liability of the epidermis to endopolyploidization. Independently of endopolyploidy the basic number of plastids (i.e., the amount reached in a cell type under given conditions, but without endopolyploidy) was altered in certain trisomes: it was higher (with extra chromosomes III through VI) or lower (II, perhaps also I) than in eudiploid control plants, the change taking the same direction in epidermal and in spongy parenchyma cells as in guard cells.     

Variations in endopolyploidy level during the short period of the early growing stage in the roots and leaves of maize (<Emphasis Type="Italic">Zea mays</Emphasis>) seedlings

We used a flow cytometer to investigate the variations in endopolyploidy (the frequencies of nuclei with DNA contents equivalent to 4C through 16C) during the short period of the early growing stage in vigorously growing young tissues of maize seedlings. We examined different portions of the root and leaves that had been growing for 7 (day 7) and 13 (day 13) days after germination. Endoreplication showed two opposing phenomena without aging. In one case, the endopolyploidy of the first leaf was higher on day 13 than on day 7. In the latter case, endopolyploidy decreased, as clearly revealed by a comparison of the endopolyploidy of the second leaves and the 160–170 mm portion of the seminal root on days 7 and 13. Endopolyploidy was also lower in the top of the leaf. In roots, endopolyploidy was increased by the exogenous application of abscisic acid for only 1 day. The levels of endopolyploidy increased without an increase in cell size in the roots. These results showed that endoreplication occurs in actively growing and young tissue and that the variation can be induced in the short period examined.     

An Experimental Study of Cell and Nuclear Growth and Their Relation to Cell Diversification within a Plant Tissue

Roots of Zea mays were grown for four days in a solution of colchicine that inhibits cell division. During this period the amount of cell growth and nuclear DNA replication was measured in different regions of the root cap. The rates at which cell volume and nuclear DNA content double are similar in the first cell-tier of the cap (the meristematic stem-cell) but in more distal cells the rate of cell growth outstrips the rate of nuclear DNA increase. It is suggested that the degree of coordination between cell and nuclear growth regulates meristematic activity and can influence the onset of cell differentiation.
The pattern of endopolyploidy changes in the different regions of the colchicine-treated root cap. It is suggested that the degree of endopolyploidy normally characteristic of cells at particular locations within this tissue is not a response to a positional signal alone; it is more likely to be due to some rate-limiting control of DNA synthesis.     

ON THE CHROMOSOMAL BASIS FOR CELLULAR DIFFERENTIATION

The relationship of somatic polyploidy to cellular differentiation is discussed. Evidences for the occurrence of endopolyploidy on the basis of auxin-induced divisions are questioned and considered valid in the light of radioisotope studies of DNA synthesis. The non-universality of endopolyploidy is shown by known exceptions. The occurrence of endopolyploidy in varying degrees in gametophytes of the same species of fern which are of different basic chromosome numbers shows its variability within a given morphogenetic pattern. From these evidences, and from an analysis of the separate component processes of cell multiplication, it can be concluded that polyploidy is not a necessary precursor or concomitant of cell differentiation, but rather is a type of differentiation in itself. As such, it may be one of a cumulative set of steps to yet other differentiation, but this may be incidental to the intrinsic nature and role of the nucleus.     

Endopolyploidy: Towards an understanding of its biological significance

There is a certain measure of perplexity concerning the significance of endopolyploidy. It seems that this results from a narrow frame of reference from which investigators view the phenomenon; that is, a predilection for emphasizing the specialized functional aspect of endopolyploidy as it operates in species at the present time overrides any consideration of the rôle that this state may play in the life of a species in its encounter with the forces of natural selection either in the past or in the future.There does not seem to be any obvious relationship between the degree of endopolyploidy that a species can exhibit and either its basic DNA content or the structure of its nucleus. The significance of endopolyploidy may reside not so much in any specialized function that the condition can support, but rather in the properties that are consequent upon the endopolyploid condition itself and which are distinct from those that apply to diploid cells. Some of the properties of the endopolyploid state, and examples of their manifestation in plants and animals, are discussed. The conclusion is that these properties have a potential that opens possibilities for new paths of development and serves as a factor upon which natural selection can operate.     

Biological role of endoreplication in the process of spermatogenesis inChara vulgaris L.

Summary During cell division in antheridial filaments ofChara vulgaris an increase in DNA content occurs in both shield cells and manubria within an antheridium, reaching 16C–64C and 8C–32C levels, respectively. Endoreplication ceases prior to the formation of spermatids and initiation of spermiogenesis, probably as a result of symplasmic isolation of the antheridium from the thallus. As the DNA content of the nuclei increases, the shield cells³H-leucine incorporation increases, and they grow intensively in the tangential plane. Translation decreases considerably after termination of shield cell growth. DNA content of mature manubria is half of that in shield cells, although their size is 10 times that of manubria. Translational activity of manubria also increases as DNA content rises and cells grow. However, during spermiogenesis, this activity remains at its maximum, which is associated with the secretory function of the manubria. Spermiogenesis is also accompanied by far-reaching ultrastructural changes within the manubrial cytoplasm.The level of endopolyploidy in both shield cells and manubria of antheridia formed in the spring is higher by one replication cycle, than in autumnal antheridia. AMO-1618, at a concentration of 10^–5M reduces the DNA content in the autumnal manubria. The higher the manubrial level of endopolyploidy in spermiogenesis, the greater their size, and the higher the translational activity and number of joined spermatids. The number of spermatozoids in the antheridium is also positively correlated with the internal volume of an antheridium, which is itself dependent on the endopolyploidy level of shield cells.The results obtained confirm the assumption that endoreplication favours the higher growth dynamics and potential translational activity, which occurs in the dynamic growth phase only in shield cells, while in manubria, i.e. cells producing substances necessary to spermatozoids development, it remains high until the end of spermiogenesis.     

Genome size, cell size, and the evolution of enucleated erythrocytes in attenuate salamanders

Within the salamander family Plethodontidae, five different clades have evolved high levels of enucleated red blood cells, which are extremely unusual among non-mammalian vertebrates. In each of these five clades, the salamanders have large genomes and miniaturized or attenuated body forms. Such a correlation suggests that the loss of nuclei in red blood cells may be related, in part, to the interaction between large genome size and small body size, which has been shown to have profound morphological consequences for the nervous and visual systems in plethodontids. Previous work has demonstrated that variation in both the level of enucleated cells and the size of the nuclear genome exists among species of the monophyletic plethodontid genus Batrachoseps. Here, we report extensive intraspecific variation in levels of enucleated red blood cells in 15 species and provide measurements of red blood cell size, nucleus size, and genome size for 13 species of Batrachoseps. We present a new phylogenetic hypothesis for the genus based on 6150 bp of mitochondrial DNA sequence data from nine exemplar taxa and use it to examine the relationship between genome size and enucleated red blood cell morphology in a phylogenetic framework. Our analyses demonstrate positive direct correlations between genome size, nucleus size, and both nucleated and enucleated cell sizes within Batrachoseps, although only the relationship between genome size and nucleus size is significant when phylogenetically independent contrasts are used. In light of our results and broader studies of comparative hematology, we propose that high levels of enucleated, variably sized red blood cells in Batrachoseps may have evolved in response to rheological problems associated with the circulation of large red blood cells containing large, bulky nuclei in an attenuate organism.     

DNA-Feulgen cytophotometric determination of genome size for the freshwater-invading copepod Eurytemora affinis.

Variation in nuclear DNA content within some eukaryotic species is well documented, but causes and consequences of such variation remain unclear. Here we report genome size of an estuarine and salt-marsh calanoid copepod, Eurytemora affinis, which has recently invaded inland freshwater habitats independently and repeatedly in North America, Europe, and Asia. Adults and embryos of E. affinis from the St. Lawrence River drainage were examined for somatic cell DNA content and the presence or absence of embryonic chromatin diminution, using Feulgen-DNA cytophotometry to determine a diploid or 2C genome size of 0.6-0.7 pg DNA/cell. The majority of somatic cell nuclei, however, have twice this DNA content (1.3 pg/nucleus) in all of the adults examined and possibly represent a population of cells arrested at the G2 stage of the cell cycle or associated with some degree of endopolyploidy. Both suggestions contradict assumptions that DNA replication does not occur in adult tissues during the determinate growth characteristic of copepods. Absence of germ cell nuclei with markedly elevated DNA values, commonly found for species of cyclopoid copepods that show chromatin diminution, indicates that E. affinis lacks this trait. The small genome size and presumed absence of chromatin diminution increase the potential utility of E. affinis as a model for genomic studies on mechanisms of adaptation during freshwater invasions.     

Hepatic endopolyploidy as a cellular consequence of age-specific selection for rate of development in mice

Endopolyploidy is the generation of polyploid cells by DNA replication without subsequent cell division and is correlated with hypertrophic growth or growth via cell size. Thus, selection that alters growth may also change onset and frequency of endopolyploidy as a correlated response. We search for endopolyploidy in the liver in response to age-specific restricted index selection for the rate of development. Polyploidy changes over ontogeny are described in five mouse lines: two selected for divergence in early growth (0-10 days of age), two selected for divergence in late growth (28-56 days of age), and one randombred control. Polyploid cell frequency within each line increased as ontogeny continued, as expected from previous research. However, selection for altered growth clearly plays a role in the frequency and onset of polyploid cells. Lines selected for divergence in early growth have polyploidy differences after weaning that are not seen in adult mice. However, lines selected for divergence in late growth are divergent in frequency of polyploid cells, starting near sexual maturity and continuing into adulthood.     

The influence of experimentally induced polyploidy on the relationships between endopolyploidy and plant function in Arabidopsis thaliana

Whole genome duplication, leading to polyploidy and endopolyploidy, occurs in all domains and kingdoms and is especially prevalent in vascular plants. Both polyploidy and endopolyploidy increase cell size, but it is unclear whether both processes have similar effects on plant morphology and function, or whether polyploidy influences the magnitude of endopolyploidy. To address these gaps in knowledge, fifty‐five geographically separated diploid accessions of Arabidopsis thaliana that span a gradient of endopolyploidy were experimentally manipulated to induce polyploidy. Both the diploids and artificially induced tetraploids were grown in a common greenhouse environment and evaluated with respect to nine reproductive and vegetative characteristics. Induced polyploidy decreased leaf endopolyploidy and stem endopolyploidy along with specific leaf area and stem height, but increased days to bolting, leaf size, leaf dry mass, and leaf water content. Phenotypic responses to induced polyploidy varied significantly among accessions but this did not affect the relationship between phenotypic traits and endopolyploidy. Our results provide experimental support for a trade‐off between induced polyploidy and endopolyploidy, which caused induced polyploids to have lower endopolyploidy than diploids. Though polyploidy did not influence the relationship between endopolyploidy and plant traits, phenotypic responses to experimental genome duplication could not be easily predicted because of strong cytotype by accession interactions.     

Ploidy mosaics: does endopolyploidy in explants affect the cytogenetic stability of orchids regenerated from PLBs?

The induction and regeneration of protocorm-like bodies (PLBs) is a morphogenetic pathway widely used for orchid micropropagation. As endopolyploidy, i.e., the coexistence of cells with different ploidy levels, is a common feature in orchid tissues, a natural question arises when using somatic tissues as explants for orchid micropropagation: does endopolyploidy in explants affect the cytogenetic stability of regenerated plantlets? To answer this question, Epidendrum fulgens was used as a model plant, and flow cytometry was used to analyze endopolyploidy in pollinia, petals, labella, leaf bases, leaf tips, root tips, and protocorm bases and apices, which were subsequently used as explants for PLB induction and plant regeneration. Ploidy screenings showed contrasting ploidy patterns in samples, endopolyploidy being detected in all tissues, with C-values ranging from 1 to 16C. Protocorm bases and root tips presented the highest proportion of endopolyploidy, while petals and protocorm apices showed the lowest proportion. Flower parts exhibited high oxidation for PLB induction and pollinia failed to produce PLB or callus. The highest induction rate occurred at 10 µM TDZ, with 92%, 22%, and 0.92% for protocorm bases, leaves, and root tips, respectively. Plantlets were more easily regenerated from PLBs induced from protocorm bases than from leaves and roots. Doubled ploidy levels were registered in a proportion of 11% and 33% for PLB-regenerated plantlets obtained from protocorm bases and leaf bases, respectively, which was not directly associated with the proportion of endopolyploid cells or cycle value of explants.

     

Estimation of nuclear DNA content in plants using flow cytometry

Flow cytometry (FCM) using DNA-selective fluorochromes is now the prevailing method for the measurement of nuclear DNA content in plants. Ease of sample preparation and high sample throughput make it generally better suited than other methods such as Feulgen densitometry to estimate genome size, level of generative polyploidy, nuclear replication state and endopolyploidy (polysomaty). Here we present four protocols for sample preparation (suspensions of intact cell nuclei) and describe the analysis of nuclear DNA amounts using FCM. We consider the chemicals and equipment necessary, the measurement process, data analysis, and describe the most frequent problems encountered with plant material such as the interference of secondary metabolites. The purpose and requirement of internal and external standardization are discussed. The importance of using a correct terminology for DNA amounts and genome size is underlined, and its basic principles are explained.