Polyploidy: significance for cardiomyocyte function and heart aerobic capacity

Somatic polyploidy, defined as genome multiplication, was found in all differentiated mammalian tissues. The highest level of such a polyploidy was found in the myocardium. This phenomenon was shown to be associated with changes in the pattern of gene expression. Hence, polyploidization may create cells with new physiology. The effect of polyploidy on the heart function has never been studied. The aim of the present study was to investigate the effect of polyploidy on cardiomyocyte functioning and heart aerobic capacity. DNA and the total protein content, nucleolar activity reflecting the rate of rRNA synthesis and, consequently, ribosome biogenesis, were measured in ventricular myocytes isolated from the human and from 21 mammalian species by image cytometry and microscopic morphometry. The total protein content was estimated after staining slides with naphtol-yellow dye. For measurement of DNA and nucleolar area, staining with Hoechst and AgNO3 was applied. Cardiac aerobic capacity was evaluated by the heart mass to body mass ratio. A negative correlation between the heart index and the average cell ploidy was revealed (r = -0.79; P < 0.0001). The average genome number per myocyte was registered to be higher by approximately 35% in the sedentary mammals, with the heart index about 0.4% from body mass, than in the athletes with heart index about 0.6% of body mass. Polyploidization was shown to be associated with a sharp decrease in the protein/DNA ratio in cardiomyocytes. As a result, cardiomyocytes in the athletic mammals with poorly polyploid hearts have much higher protein content per genome than do cells in the sedentary species with highly polyploid hearts. Surprisingly, despite decreased protein/DNA ratio, the nucleolar area per genome significantly increased with polyploidization, indicating the imbalance between the cellular protein content and the rate of ribosome biogenesis. Such an imbalance should obviously impair cardiac function, because the additional genomes take some valuable space and biological resources from the cell, which could have been otherwise directed to the maintenance of cardiomyocyte contractile machinery. It is generally accepted that somatic polyploidy is associated with oxidative stress and energetic starvation. Thus, we suppose that additional genomes may serve for cardiomyocyte protection from oxidative damage in the hearts.     

Growth of cardiac muscle cells during rat adaptation to altitude hypoxia

The weight of the right heart ventricle in 1.5-month-old rats kept after birth in the mountains of 3400 m altitude is higher and its muscle cell cytoplasm mass is much larger compared to those in 1.5-month-old animals raised at 800 m altitude. The hypertrophy of cells is not due to their polyploidization. Only a small increase in the relative number of polyploid cells takes place under high altitude hypoxia. The weight of the right ventricle and myocyte mass in 3-month-old rats kept 1.5-3 months after the birth at 3400 m altitude also increases, although this augmentation is significantly less than in the animals grown in the mountains for 1.5 months immediately after the birth. The myocyte ploidy of adult animals adapted to hypoxia does not essentially differ from that of 1.5- and 3-month-old control rats: about 80 per cent of these cells are polyploid. Thus, the growth of cardiac myocytes under the heart hyperfunction in the case of high altitude hypoxia proceeds mainly on the ground of the stable polyploid genome, as well as normal ontogenetic growth of these cells.     

Cardiomyocyte ploidy levels in birds with different growth rates

Cytofluorimetric study of ploidy levels in ventricular cardiomyocytes was carried out on 36 adult bird species belonging to 10 orders as well as on the quail Coturnix coturnix, of different ages. It was shown that polyploidization of quail cardiomyocytes occurs during the first 40 days after hatching and ends by the time growth is completed. In adult birds, the cardiomyocyte ploidy hardly changed at all. Interspecies comparison revealed that in the adult bird myocardium 2cx2 myocytes are predominant, accounting for at least 50% of the cell population. Multinuclear cells with three to eight diploid nuclei were widespread. The percentage of such cells was five to six times higher in precocial species than in altricial birds of the same weight. Myocytes with polyploid nuclei were rare. A significant interspecies variability of cardiomyocyte ploidy levels was observed. The most prominent differences were found between the precocial and the altricial birds. The mean number of genomes in cells correlated both with the body mass and with the growth rate of the birds. The differences between the precocial and altricial birds disappeared when a statistical method was used to eliminate the effect of the growth rate, but did not when the effect of body mass was eliminated. Among the altricial birds, which are generally immobile during growth, the cardiomyocyte ploidy levels also correlated more closely with growth rate than with body mass. The opposite was observed in the precocial birds, which are highly mobile from the first minutes of life. We conclude that the interspecies variability of bird cardiomyocyte ploidy levels is a result of changes in the balance between the cardiac functional load and the growth rate; this is manifested at the cellular level as a competition between the proliferation and differentiation of cardiomyocytes. J. Exp. Zool. 289:48-58, 2001.     

Heart and liver as developmental bottlenecks of mammal design: evidence from cell polyploidization

A new concept in physiological evolution in mammals is proposed based on the data of cell and developmental biology. The levels of mammal cardiomyocyte and hepatocyte ploidy were analysed with regards to body mass, growth rate in different periods of ontogenesis, and maximal functional potential of the heart and liver in the adult state. For both organs, cell ploidy and functional potential of the organ correlated inversely. Polyploid cells were shown to have a lower amount of protein per genome and a lower ratio of mitochondrial internal membrane to tissue‐specific cellular machinery (i.e. a lower energy supply) compared with diploid cells. Both cell ploidy and functional potential of the organ correlated with the animal growth rate that controls the organ functional load, but only if measured during the period of ontogenesis when polyploidization of the particular organ proceeds. However, correlation was in opposite directions: negative in the heart and positive in the liver. This is because these organs provide predominantly for the antagonistic tasks that they perform during ontogenesis. The liver provides a gain in body mass, whereas the heart maintains locomotor activity. The balance between these tasks depends on the growth rate constrained by the degree of precocity of development. During ontogenesis, an organ that works intensively in the adult state is subjected to a low workload and is furnished plentifully by resources. On the contrary, an organ with a low functional potential in the adult state starts to work intensively just after birth and experiences a shortage of resources during growth. Our data indicate that somatic polyploidization occurs as a result of tension between growth and function caused by the limitation of resources. The highest levels of mammal cell ploidy occur in the heart and the liver, although in different species which are positioned on the opposite ends of the growth rate range. Thus, the heart and the liver seem to be developmental bottlenecks of mammal design, determining the limits of metabolic capacity of adult animals. Compared with other mammals, the human has a very slow growth rate and a very high ploidy of cardiomyocytes (thus confirming the revealed regularity). This helps explain the well‐known fact that cardiovascular failure is the number one cause of human mortality. © 2004 The Linnean Society of London, Biological Journal of the Linnean Society, 2004, 83 , 175–186.     

Myocyte ploidy in heart chambers of birds with different locomotor activity

The ploidy levels of atrio- and ventriculocytes were determined by means of cytofluorimetry in 31 species of birds. The obtained data were collated with postnatal growth rate, heart mass index, and relative masses of heart chambers. The difference between mean ploidy of cardiomyocytes in the left and right atrium is small (7.9+/-0.6%) and comparable to the difference in the masses of these chambers (10.5+/-0.8%). The difference between mean ploidy of atrio- and ventriculocytes is most pronounced for the left and right parts of heart (23.9+/-1.4% and 24.0+/-1.3%, respectively) and corresponds to considerable differences in the average masses of atria and ventricles (4.5-fold and 2.1-fold, respectively). The mean cardiomyocyte ploidy levels in the left and right ventricles differ only slightly, as in the case of atria (by 8.1+/-0.5%), whereas the average mass of the left ventricle is greater by 237+/-16%. This discord can be explained by peculiarities of the growth, which is nonproportionally faster in the left ventricle during the last stage of proliferative heart growth as compared to other chambers. The cardiomyocyte ploidy is higher in birds with a relatively small heart and lower ability to flight. Birds with a high locomotor activity in the adult state have an athletic heart (mass index >1%); they are fast growing, altricial species with a low heart workload in the early postnatal ontogenesis. Birds with a low locomotor activity at the adult state are precocial; they grow slowly and have a high locomotor activity from the first minutes of life. Thus, notwithstanding the fact that a greater elevation of cardiomyocyte ploidy level is acquired under a higher functional load (ventricles vs. atria, left vs. right part of the heart), it is associated with a lower functional potential of the organ at the adult state. The level of somatic polyploidy can be considered an indicator of developmental tensions arising due to a high workload during the growth of a given organ and deficiency of resources invested into this growth. J. Exp. Zool. 293:427-441, 2002.     

Association of ploidy and sexual system in Lycium californicum (Solanaceae)

In North American Lycium (Solanaceae), the evolution of gender dimorphism has been proposed as a means of restoring outcrossing after polyploidization causes the loss of self-incompatibility. Previous studies of this process in Lycium focused on comparisons between species that differ in ploidy. We examined intraspecific variation in floral morphology and DNA content in populations of L. californicum to determine correlations between sexual system and cytotype. We also used nuclear ITS and GBSSI sequence data to determine whether diploid and polyploid forms represent the same phylogenetic species, and the phylogeographic relationships among populations and ploidy levels. Within populations, no variation in ploidy was found, although among populations there was a perfect correspondence between sexual system and cytotype. Diploid populations were all hermaphroditic, whereas tetraploid populations were all gender dimorphic. There was no clear geographic pattern to the occurrence of diploid and tetraploid forms. Phylogenetic analysis confirms that L. californicum, regardless of ploidy, forms a monophyletic group within the genus Lycium. Sequences from diploid and polyploid individuals did not form reciprocally monophyletic clades, indicating either multiple gains of polyploidy, ongoing gene flow between cytotypes, or lack of lineage sorting since the evolution of polyploidy. The correspondence between ploidy and sex expression is consistent with the hypothesis that polyploidization triggers the evolution of gender dimorphism in this and other Lycium species.     

Cellular aspects of pathogenesis of hypertrophic cardiomyopathy: Role of cardiomyocyte polyploidy and activation of nuclear antigen of the proliferating cell in myocardium

For a comparative analysis of cytomorphological characteristics of hypertrophied interventricular septum (IVS), both patients different ages with severe courses of obstructive hypertrophic cardiomyopathy (OHCMP) were examined, including children, and patients with essential arterial hypertension (EAH). The course of OHCMP in children as compared with adults was found to be characterized by considerable IVS hypertrophy that was accompanied by an acceleration of cardiomyocyte polyploidization. The mean ploidy level of cardiomyocytes in children with OHCMP was higher than in adult patients. The mean ploidy level of nuclei, the number of prolipherative cell nuclear antigen (PCNA)-positive nuclei, and the number of polyploid cardiomyocyte nuclei in adult patients with OHCMP were significantly higher statistically than in patients with EAH. The PCNA-positive labels in stromal cells were revealed only in patients with OHCMP. The obtained data indicate an important role of cardiomyocyte polyploidy and of activation of the proliferating cell nuclear antigen in development of myocardial hypertrophy in patients with OHCMP.     

Effects of arbuscular mycorrhizal colonization and phosphorus application on nuclear ploidy in Allium porrum plants

Arbuscular mycorrhizal (AM) colonization can strongly affect the plant cell nucleus, causing displacement from the periphery to the center of the cell, hypertrophy and polyploidization. The hypertrophy response has been shown in a variety of AM plants whilst polyploidization has been reported only in Lycopersicon esculentum, a multiploid species with a small genome. In order to determine whether polyploidization is a general plant response to AM colonization, analyses were performed on Allium porrum, a plant with a large genome, which is much less subject to polyploidization than L. esculentum. The ploidy status of leaves, complete root systems and four zones of the adventitious roots was investigated in relation to phosphorus content, AM colonization and root differentiation in A. porrum plants grown under two different regimes of phosphate nutrition in order to distinguish direct effects of the fungus from those of improved nutrition. Results showed the presence of two nuclear populations (2C and 4C) in all treatments and samples. Linear regression analyses suggested a general negative correlation between phosphorus content and the proportion of 2C nuclei. The percentage of 2C nuclei (and consequently that of 4C nuclei), was also influenced by AM colonization, differentiation and ageing of the root cells, which resulted in earlier occurrence, in time and space, of polyploid nuclei.     

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.     

Ploidy in cyanobacteria

A recently developed real-time PCR method for the determination of genome copy numbers was optimized for the application to cyanobacteria. Three species were chosen to represent a fresh water species, a salt water species, and two strains of a widely used laboratory species. Synechococcus PCC 7942 and Synechococcus WH7803 were found to contain 3-4 genome copies per cell and are thus oligoploid, confirming earlier publications. In contrast, Synechocystis PCC 6803 is highly polyploid. The motile wild-type strain contains 218 genome copies in exponential phase and 58 genome copies in linear and in stationary growth phase. The GT wild-type strain contains 142 genome copies in exponential phase and 42 genome copies in linear and stationary growth phase. These are the highest numbers found for any cyanobacterial species. Notably these values are much higher than the value of 12 genome copies published for the 'Kazusa' strain more than 20 years ago. The results reveal that for Synechocystis PCC 6803 strain differences exist and that the ploidy level is highly growth phase-regulated. A compilation of the ploidy levels of all investigated cyanobacterial species gives an overview of the genome copy number distribution and shows that monoploid, oligoploid, and polyploid cyanobacteria exist.     

Relationship of hepatocyte ploidy levels with body size and growth rate in mammals.

To elucidate possible causes of the elevation of genome number in somatic cells, hepatocyte ploidy levels were measured cytofluorimetrically and related to the organismal parameters (body size, postnatal growth rate, and postnatal development type) in 53 mammalian species. Metabolic scope (ratio of maximal metabolic rate to basal metabolic rate) was also included in 23 species. Body masses ranged 10(5) times, and growth rate more than 30 times. Postnatal growth rate was found to have the strongest effect on the hepatocyte ploidy. At a fixed body mass the growth rate closely correlates (partial correlation analysis) with the cell ploidy level (r = 0.85, P < 10(-6)), whereas at a fixed growth rate body mass correlates poorly with ploidy level (r = -0.38, P < 0.01). The mature young (precocial mammals) of the species have, on average, a higher cell ploidy level than the immature-born (altricial) animals. However, the relationship between precocity of young and cell ploidy levels disappears when the influences of growth rate and body mass are removed. Interspecies variability of the hepatocyte ploidy levels may be explained by different levels of competition between the processes of proliferation and differentiation in cells. In turn, the animal differences in the levels of this competition are due to differences in growth rate. A high negative correlation between the hepatocyte ploidy level and the metabolic scope indicates a low safety margin of organs with a high number of polyploid cells. This fact allows us to challenge a common opinion that increasing ploidy enhances the functional capability of cells or is necessary for cell differentiation. Somatic polyploidy can be considered a "cheap" solution of growth problems that appear when an organ is working at the limit of its capabilities.     

Upregulation of Nox4 in the aging vasculature and its association with smooth muscle cell polyploidy

Our recent reports indicated that polyploidization of aortic vascular smooth muscle cells (VSMC) serves as a biomarker for aging, and that the polyploid state is linked to a higher incidence of senescence in vivo. Here, we found that NADPH oxidase 4 (Nox4) expression is augmented in VSMC from aortas of old rats and that Nox4 levels are increased in polyploid VSMC in comparison to diploid cells in vivo. Seeking to determine if Nox4 upregulation plays a causal role in the accumulation of polyploid cells, we performed ploidy analysis on primary VSMC transduced with Nox4 adenovirus. We observed a consistent accumulation of polyploid cells and a concomitant decrease in the percentage of diploid cells in Nox4 overexpressing cells in comparison to controls or to cells overexpressing dominant negative Nox4. Further exploration of this phenomenon in VSMC cultures identified a Nox4-induced decrease in the chromosome passenger protein, survivin, whose absence and mislocalization during polyploidization was previously shown to induce VSMC polyploidy. Taken together, our study is the first to show increased Nox4 levels in VSMC during aging, and to demonstrate its role in induction of polyploidy in this lineage.     

FISH mapping of 35S and 5S rRNA genes in Artemisia subgenus Dracunculus (Asteraceae): changes in number of loci during polyploid evolution and their systematic implications

Fluorescence in situ hybridization (FISH) with 35S and 5S rDNA probes was used to characterize cytogenetically representatives of Artemisia subgenus Dracunculus and allied species and to explore their evolution following polyploidization. At the diploid level two rDNA loci were observed in most species belonging to the A. dracunculus complex, a pattern considered to be the ancestral state for diploid Artemisia. In contrast, representative species from the Eurasian grade which belong to the other major lineage of the subgenus had more heterogeneous rDNA profiles, with three to five loci at the diploid level. Divergent patterns of locus evolution were also detected in polyploids, with the number and distribution of rDNA loci broadly fitting the two main diversification lineages in the subgenus. In the polyploid complex of A. dracunculus, the number of rDNA loci was almost proportional to ploidy, although monoploid genome size was shown to decrease with increasing ploidy. However, in polyploids from the Eurasian grade we found a remarkable reduction in the number of rDNA sites, suggesting that these species might have experienced either a complete loss of loci or a significant reduction in the number of repeats following polyploid formation. © 2013 The Linnean Society of London, Biological Journal of the Linnean Society, 2013 , 171 , 655–666.     

Genetic response of Paspalum plicatulum to genome duplication

Paspalum plicatulum is a perennial rhizomatous grass with natural diploid and polyploid cytotypes. In this study, we investigated the occurrence of sequence polymorphisms arising immediately after genome autoduplication in this species. Two mixoploid plants (4C and 7D) were previously obtained through colchicine treatment of seeds generated by open pollination of a diploid plant (H14-2x). Diploid and tetraploid sectors from both mixoploids were dissected to generate two ploidy series (4C-2x/4C-4x and 7D-2x/7D-4x). Molecular fingerprints were generated from the maternal plant H14-2x, both ploidy series (4C-2x/4C-4x and 7D-2x/7D-4x), and a tetraploid plant (C1) produced by selfing 7D-4x. Our results indicate that immediately after polyploidization P. plicatulum suffers genetic rearrangements affecting ~28–38 % of the genome. Band gain and loss were equally prevalent at a statistically significant level. At least 5.62 % of the genome experimented recurrent genetic variation in a non-random basis with a confidence of 94.88 %. A significant proportion of novel bands (36 out of 195; 18.4 %) was detected in the C1 tetraploid plant. Half of these bands were not amplified in either H14-2x or 7D-4x, while the remainders were present in H14-2x but absent in 7D-4x. Our results indicate the occurrence of a considerable number of genetic changes in P. plicatulum immediately after polyploidization, some of which were recurrently detected in different independent events. Moreover, we confirmed that after polyploidization, lost ancestral alleles were spontaneously recovered in further generations, a phenomenon previously reported by other research groups.     

Morphofunctional parameters of the nucleoli during development of polyploid nutrient cells of gonads from the snail Succinea lauta

Ag-protein contents, integral area and number of nucleoli in polyploidizing nuclei of gonadal nutrient cells of the snail Succinea lauta were estimated on the squashed preparations by means of morphometry and cytophotometry. 8 NORs of different size were found in haploid chromosome set of prophase spermatocytes (n = 22), but usually 1-2 nucleoli per 2c DNA are present in the nutrient cell nuclei. During genome multiplication from 2c to 32c-64c the Ag-proteins content of nucleoli increased proportionally to gene dosage, but irregularly: before 8c-level the coefficient of increasing in each endocycle was more than 2; from 8c to 16c it was 2; after 16c-level it usually decreased to 1.6-1.3. This dynamics reflects the effects of several factors on nucleolar activity: endomitotic polyploidy (gene dosage effect), differentiation and rhythmic functioning of tissue. Increasing indexes of integral area and the number of nucleoli during polyploidization were significantly less, than increasing index of Ag-proteins. The lag of nucleolar area for 4 cycles (2c-32c) was 32%, and number of nucleoli per diploid set decreased from 2 to 1. It may be due to NOR aggregation corresponding chromosomes. The photometric index of Ag-protein content more adequately reflects in the nucleolar activity during development and functioning of tissues.     

Genome copy numbers and gene conversion in methanogenic archaea

Previous studies revealed that one species of methanogenic archaea, Methanocaldococcus jannaschii, is polyploid, while a second species, Methanothermobacter thermoautotrophicus, is diploid. To further investigate the distribution of ploidy in methanogenic archaea, species of two additional genera-Methanosarcina acetivorans and Methanococcus maripaludis-were investigated. M. acetivorans was found to be polyploid during fast growth (t(D) = 6 h; 17 genome copies) and oligoploid during slow growth (doubling time = 49 h; 3 genome copies). M. maripaludis has the highest ploidy level found for any archaeal species, with up to 55 genome copies in exponential phase and ca. 30 in stationary phase. A compilation of archaeal species with quantified ploidy levels reveals a clear dichotomy between Euryarchaeota and Crenarchaeota: none of seven euryarchaeal species of six genera is monoploid (haploid), while, in contrast, all six crenarchaeal species of four genera are monoploid, indicating significant genetic differences between these two kingdoms. Polyploidy in asexual species should lead to accumulation of inactivating mutations until the number of intact chromosomes per cell drops to zero (called "Muller's ratchet"). A mechanism to equalize the genome copies, such as gene conversion, would counteract this phenomenon. Making use of a previously constructed heterozygous mutant strain of the polyploid M. maripaludis we could show that in the absence of selection very fast equalization of genomes in M. maripaludis took place probably via a gene conversion mechanism. In addition, it was shown that the velocity of this phenomenon is inversely correlated to the strength of selection.     

PIDDosome‐induced p53‐dependent ploidy restriction facilitates hepatocarcinogenesis

Polyploidization frequently precedes tumorigenesis but also occurs during normal development in several tissues. Hepatocyte ploidy is controlled by the PIDDosome during development and regeneration. This multi‐protein complex is activated by supernumerary centrosomes to induce p53 and restrict proliferation of polyploid cells, otherwise prone for chromosomal instability. PIDDosome deficiency in the liver results in drastically increased polyploidy. To investigate PIDDosome‐induced p53‐activation in the pathogenesis of liver cancer, we chemically induced hepatocellular carcinoma (HCC) in mice. Strikingly, PIDDosome deficiency reduced tumor number and burden, despite the inability to activate p53 in polyploid cells. Liver tumors arise primarily from cells with low ploidy, indicating an intrinsic pro‐tumorigenic effect of PIDDosome‐mediated ploidy restriction. These data suggest that hyperpolyploidization caused by PIDDosome deficiency protects from HCC. Moreover, high tumor cell density, as a surrogate marker of low ploidy, predicts poor survival of HCC patients receiving liver transplantation. Together, we show that the PIDDosome is a potential therapeutic target to manipulate hepatocyte polyploidization for HCC prevention and that tumor cell density may serve as a novel prognostic marker for recurrence‐free survival in HCC patients.     

S6K1 is involved in polyploidization through its phosphorylation at Thr421/Ser424

Studies on polyploidization of megakaryocytes have been hampered by the lack of synchronized polyploid megakaryocytes. In this study, a relatively synchronized polyploid cell model was successfully established by employing Dami cells treated with nocodazole. In nocodazole‐induced cells, cyclin B expression oscillated normally as in diploid cells and polyploid megakaryocytes. By using the nocodazole‐induced Dami cell model, we found that 4E‐BP1 and Thr421/Ser424 of ribosomal S6 kinase 1(S6K1) were phosphorylated mostly at M‐phase in cytoplasm and oscillated in nocodazole‐induced polyploid Dami cells, concomitant with increased expression of p27 and cyclin D3. However, phosphorylation of 4E‐BP1 and S6K1 on Thr421/Ser424 was significantly decreased in differentiated Dami cells induced by phorbol 12‐myristate 13‐acetate (PMA), concomitant with increased expression of cyclin D1 and p21 and cyclin D3. Overexpression of the kinase dead form of S6K1 containing the mutation Lys 100 → Gln in PMA‐induced Dami cells increased ploidy whereas overexpression of rapamycin‐resistant form of S6K1 containing the mutations Thr421 → Glu and Ser424 → Asp significantly dephosphorylated 4E‐BP1 and reduced expression of cyclin D1, cyclin D3, p21 and p27, and slightly decreased the ploidy of PMA‐induced Dami cells, compared with treatment with PMA alone. Moreover, overexpression of rapamycin‐resistant form of S6K1 significantly reversed polyploidization of nocodazole‐induced Dami cells. Furthermore, MAP (a novel compound synthesized recently) partly blocked the phosphorylation of S6K1 on Thr421/Ser424 and decreased the expression of p27 and polyploidization in nocodazole‐induced Dami cells. Taken together, these data suggested that S6K1/4E‐BP1 pathway may play an important role in polyploidization of megakaryocytes. J. Cell. Physiol. 219: 31–44, 2009. © 2008 Wiley‐Liss, Inc.     

Phenotypic and gene expression analyses of a ploidy series of maize inbred Oh43

Polyploidization has repeatedly occurred during plant evolution. Although autopolyploidy is the best model to characterize the polyploidization effects in a highly controlled manner, there are limited studies on autopolyploids compared to allopolyploids. To improve our understanding of autopolyploidy effects in maize, we developed an inbred Oh43 ploidy series consisting of the diploid (2X), tetraploid (4X) and hexaploid (6X) lines and compared their phenotypes and gene expression in the mature adult leaf tissue. Our phenotypic study showed that plants of higher ploidy exhibit increased cell size but slower growth rate, later flowering, fewer tassel branches, reduced stature and fertility. Two-dimensional difference gel electrophoresis (2D DIGE) and gel electrophoresis followed by liquid chromatography and mass spectrometry (GeLC-MS) assays of the leaf proteomes revealed ~40 and 26% quantitative differentially expressed (DE) proteins, respectively, at the per genome level. A small number of qualitative DE proteins were also identified in the GeLC-MS assay. The majority of the quantitative DE proteins found in the 2D DIGE assay were present in either the 4X versus 6X or the 2X versus 6X comparison but not the 2X versus 4X comparison. Aneuploidy in some 6X plants might contribute to the more extensive changes of gene expression per genome in the 6X. Most changes of the protein expression per genome are less than twofold. Less than 5% of the DE genes exhibit a positive or negative continuous correlation through the ploidy series between their protein expression per genome, and the genome copy number. Hence, in the Oh43 ploidy series, expression for most proteins in a cell increases linearly with ploidy.