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

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

Chromosome endoreduplication in endosperm cells of two maize genotypes and their progenies

Summary Chromosome endoreduplication is a very common process in higher plants but its function and genetic control are still to be clarified. In our experiments we analyzed, by Feulgen cytophotometry, chromosome endoreduplication in endosperm cells of two maize genotypes, IHP and ILP, having high and low protein content in their seed, respectively. Chromosome endoreduplication occurs in both lines within 24 days after pollination, attaining a maximum ploidy level of 384C (7 DNA replication rounds) in IHP and of 192C (6 replication rounds) in ILP. In the mature seed, endosperms of the two lines show different mean ploidy level. In reciprocal crosses between IHP and ILP the f₁ endosperms have mean ploidy levels analogous to that of the maternal parent, showing that the difference in ploidy level between the two genotypes is maintained. After selfing of the f₁ plants, the difference in ploidy level between the two F₂ populations is reduced. In F₂ the mean ploidy level is as variable as in f₁, indicating the absence of genetic segregation. From our data, it is apparent that both the genetic constitution (cytoplasmic and nuclear) of the maternal parent and the genotype of the individual endosperms influence the ploidy level. An analysis of the protein content in endosperms carried out on the same seed sample as analyzed cytophotometrically showed that the protein content increases, during seed development, parallel to chromosome endoreduplication and varies, in the two lines, in reciprocal crosses and their progeny, according to the same trend as mean ploidy level, suggesting a correlation between the two parameters.     

Water deficit in developing endosperm of maize: cell division and nuclear DMA endoreduplication

Water deficit severely decreases maize (Zea mays L.) kernel growth; the effect is most pronounced in apical regions of ears. The capacity for accumulation of storage material in endosperms is thought to he partially determined by the extent of cell division and endoreduplication (post-mitotic nuclear DNA synthesis). To gain a better understanding of the regulatory mechanisms involved, we have examined the effect of water deficit on cellular development during the post-fertilization period. Greenhouse-grown maize was subjected to water-limited treatments during rapid cell division [from 1 to 10days after pollination (DAP)] or rapid endoreduplication (9 to 15 DAP). The number of nuclei and the nuclear DNA content were determined with flow cytometry. Water deficit from 1 to 10 DAP substantially decreased the rate of endosperm cell division in apical-region kernels, but had little effect on middle-region endosperms. Rewatcring did not allow cell division to recover in apical-region endosperms. Water deficit from 9 to 15 DAP also decreased cell division in apical-region endosperms. Endoreduplication was not affected by the late treatment in either region of the car, but was inhibited by the early treatment in the apical region. In particular, the proportion of nuclei entering higher DN A-content size classes was reduced. We conclude that cell division is highly responsive to water deficit, whereas endoreduplication is less so. We also conclude that the reduced proportion of nuclei entering higher DNA-content size classes during endoreduplication is indicative of multiple control points in the mitotic and endoreduplication cycles.     

Inheritance of nuclear DNA content in leaf epidermal cells of Zea mays L.

 The nuclear DNA content (ploidy level) of maize leaf-epidermal cells was investigated by Feulgen cytophotometry in two lines, Illinois High Protein (IHP) and Illinois Low Protein (ILP), their reciprocal hybrids, and their F₂s. Epidermal cells have a 2C, 4C or 8C nuclear DNA content. The mean DNA content per nucleus in IHP was significantly higher than in ILP; the mean DNA content per nucleus in hybrids was intermediate between the parental lines, and the same DNA content was measured in reciprocal crosses. In F₂s the same mean DNA content as in F₁s was observed but with larger variability than in the F₁, possibly indicating genetic segregation. It is inferred that the ploidy level in the leaf epidermis is inherited, and incomplete dominance occurs in hybrids. The same behaviour in the different genotypes was observed for epidermal cell-surface area, except that an increase of mean surface area occurred in the F₁, probably due to heterotic effects. The difference in the accumulation of 4C and 8C nuclei in leaf epidermis parallels that reported between two genotypes for the endosperm tissue: to the greater chromosome endoreduplication found in the endosperm there were correspondingly higher frequencies of 4C and 8C nuclei in the leaf epidermis, indicating a higher general tendency to chromosome endoreduplication in IHP than in ILP. It is suggested that the accumulation of 4C nuclei (G₂-block) in the leaf epidermis may be regarded as the initial step of chromosome endoreduplication, the two phenomena being related to the control of the sequence DNA synthesis-mitosis, possibly involving the same genes in both endosperm and leaf. However, the inheritance of DNA content per nucleus in epidermal tissue seems to be different from that observed in endosperm tissue of the same genotypes, suggesting that differences may occur in the regulation of the activity of these genes. Received: 19 November 1996 / Accepted: 29 November 1996     

Endoreduplication of nuclear DNA in the developing maize endosperm

Multiparametric flow cytometry was used to analyze the development of the endosperm in Zea mays L. during the period from 8 to 20 days after pollination (dap). Nuclear size, DNA content per nucleus, and frequencies of nuclei with varying properties were measured in preparations that included all of the endosperm nuclei of single kernels of the inbred strain Al88. Characteristics of nuclear populations from different kernels on the same ear showed minimal variation. The dynamic changes of non-mitotic cells involved in endosperm development consisted of alternating periods of DNA replication with non-replication. Seven rounds of DNA replication had occurred in some nuclei in the later developmental stages with the rate averaging approximately one round per 24-hour period. Analysis of the DNA levels in the nuclei showed an exact doubling pattern indicating an endoreduplication process, that is, replication of the entire genome during each round. The loosely organized polytenization of the chromatin occurred to varying extents among the nuclei within an endosperm. A weak positive correlation existed between DNA content and size of nuclei suggesting that DNA increases and nuclear growth may not be highly coordinated in this tissue. Increased proportions of the larger nuclei occurred in the later stages of endosperm development. Considering the entire endosperm, the average DNA content per nucleus at the 15-dap peak level was approximately 12.8 C constituting a 2.7-fold overall increase from 8 dap.     

Experimental designs and statistical methods for mapping quantitative trait loci underlying triploid endosperm traits without maternal genetic variation

Many endosperm traits are related to grain quality in cereal crops. Endosperm traits are mainly controlled by the endosperm genome but may be affected by the maternal genome. Studies have shown that maternal genotypic variation could greatly influence the estimation of the direct effects of quantitative trait loci (QTLs) underlying endosperm traits. In this paper, we propose methods of interval mapping of endosperm QTLs using seeds of F2 or BC1 (an equal mixture of F1 x P1 and F1 x P2 with F1 as the female parent) derived from a cross between 2 pure lines (P1 x P2). The most significant advantage of our experimental designs is that the maternal effects do not contribute to the genetic variation of endosperm traits and therefore the direct effects of endosperm QTLs can be estimated without the influence of maternal effects. In addition, the experimental designs can greatly reduce environmental variation because a few F1 plants grown in a small block of field will produce sufficient F2 or BC1 seeds for endosperm QTL analysis. Simulation studies show that the methods can efficiently detect endosperm QTLs and unbiasedly estimate their positions and effects. The BC1 design is better than the F2 design.     

DNA endoreduplication in maize endosperm cells: the effect of exposure to short-term high temperature

DNA endoreduplication in Zea mays L. (cv. A619 × W64A) endosperm peaks between 16 and 18 d after pollination (DAP). The physiological function of DNA endoreduplication is not known but it is believed to be important in maize kernel development. In the present study, we investigated how 2, 4 or 6 d of high temperature (35 °C) affected DNA endoreduplication and maize kernel development in comparison with control kernels grown at 25 °C. Data were collected on fresh weight (FW), nuclei number, mitotic index, and DNA endoreduplication. Maize endosperm FW and nuclei number were reduced by exposure to 4 or 6 d of high temperature. At 18 DAP, the 2 d high temperature treatment (HTT) caused a reduction in FW and nuclei number, but had no effect on DNA endoreduplication and average DNA content per endosperm. However, when the exposure to high temperature was increased to 4 or 6 d, FW, nuclei number and the magnitude of DNA endoreduplication were progressively reduced, and the peak mitotic index was delayed compared with the control endosperm. At 18 DAP, the 4 d treatment showed 54·7% of the cells were 3 or 6 C, whereas only 41·2% were 12 C or higher. Six days of high temperature also resulted in a reduction in endosperm FW, nuclei number and a delay in the peak of mitotic index. DNA endoreduplication occurred in the kernels exposed to this treatment, although the magnitude was severely reduced compared with the control kernels. Nuclear DNA content was highly correlated (r = 0·93) with kernel FW, suggesting an important role of DNA endoreduplication in determining endosperm FW. The data suggest that high temperature during endosperm cell division exerted negative effects on DNA endoreduplication by dramatically reducing the nuclei number, leaving fewer nuclei available for DNA endoreduplication. However, the data also suggest that prolonged exposure to high temperature restricts entry of mitotic cells into the endoreduplication phase of the cell cycle.     

Genetic analyses of endoreduplication in Zea mays endosperm: evidence of sporophytic and zygotic maternal control

Flow cytometry was used to assess the variability of endoreduplication in endosperms of maize inbred lines. Little variation was found between midwestern dent types, and high levels of endoreduplication were observed in popcorns. Endoreduplication is different between inbred lines by 13-18 days after pollination, and flow cytometric analysis of ploidy level was feasible until 20 DAP. To study the genetic regulation of endoreduplication, four inbreds were crossed to B73 and developing endosperms from both parental, reciprocal F(1), and backcross generations were subjected to flow cytometric analysis. Three measurements of endoreduplication were calculated from these data and analyzed as quantitative genetic traits. Multiple models of trait inheritance were considered including triploid, diploid, sporophytic maternal, and maternal and paternal zygotic nuclear inheritance. Maternal zygotic effects, often considered a form of parental imprinting, and maternal sporophytic effects were detected. To test the feasibility of introgressing a high endoreduplication phenotype into a midwestern dent inbred line, a backcross population was generated from B73 x Sg18. Parental and progeny endoreduplication levels were compared and heritabilities assessed. The heritabilities calculated from these data generally agree with the values calculated in the larger crossing experiments.     

Cell cycle progression during endosperm development in Zea mays depends on parental dosage effects

Interploidy crosses in flowering plants often cause seed abortion. Studies in maize have shown that failure of kernel development results from dosage effects among products of imprinted but as-yet-unknown genes in the endosperm, and that the operative stoichiometry is established for a ratio of two maternal genomes to one paternal genome. In this study, we used flow cytometry to monitor cell cycle activities in developing endosperms obtained after reciprocal crosses between diploid and tetraploid maize individuals. Our data show that dosage effects alter critical events involved in the establishment of endoreduplication during maize endosperm development. Particularly, maternal genomic excess (4x x 2x crosses) forces endosperm cells to enter early into endoreduplication while paternal genomic excess (2x x 4x crosses) prevents its establishment. Our results also suggest that altering mechanisms depend on two different sets of cell cycle regulatory genes--one imprinted through the female that is required for mitotic arrest, and another responsible for re-entry into S phase that is imprinted through the male. Further, molecular and physiological analyses should provide insights into the interaction of parental imprinting action and cell cycle regulation during endosperm development.     

Plant Nucleases: VI. GENETIC AND DEVELOPMENTAL VARIABILITY IN RIBONUCLEASE ACTIVITY IN INBRED AND HYBRID CORN ENDOSPERMS

The nuclease activity of developing corn endosperms was found to consist mainly of plant RNase I during the period of major deposition of dry weight. The RNase concentrations in most inbred lines and hybrids increased throughout development, but there were large differences among genotypes in the enzyme levels at all stages. Crosses were made among inbreds classified as containing high or low RNase levels. In most cases, the general patterns of enzyme levels during development of the hybrid endosperms were not changed greatly, or showed intermediate levels of activity compared to the inbred parents. When Oh43 was used as a maternal parent, two contrasting developmental patterns were produced by using two low RNase inbreds as pollen parents. There appear to be genetic controls not only on the gross RNase levels, but also on the timing of RNase synthesis and on its stability after the cells mature. Environmental influences on RNase levels in the endosperm were noted one year.At 18 days after pollination, the RNase levels in the endosperm crown were as much as 10 times higher than in the base. By 35 days after pollination, the enzyme levels were generally uniform; at 50 days, the basal tissue usually contained the highest levels. In some genotypes, however, the enzyme levels fell in the crown while they rose in the base. These changes suggest that RNase may be associated with developmental controls that operate as the different portions of the endosperm cease cell division and begin synthesis of starch and zein.     

Nonadditive expression and parent-of-origin effects identified by microarray and allele-specific expression profiling of maize endosperm

Plant endosperm cells have a nuclear ratio of two maternal genomes to one paternal genome. This 2 to 1 dosage relationship provides a unique system for studying the additivity of gene expression levels in reciprocal hybrids. A combination of microarray profiling and allele-specific expression analysis was performed using RNA isolated from endosperm tissues of maize (Zea mays) inbred lines B73 and Mo17 and their reciprocal hybrids at two developmental stages, 13 and 19 d after pollination. The majority of genes exhibited additive expression in reciprocal hybrids based on microarray analyses. However, a substantial number of genes exhibited nonadditive expression patterns, including maternal like, paternal like, high parent like, low parent like, and expression patterns outside the range of the parental inbreds. The frequency of hybrid expression patterns outside of the parental range in maize endosperm tissue is much higher than that observed for vegetative tissues. For a set of 90 genes, allele-specific expression assays were employed to monitor allelic bias and regulatory variation. Eight of these genes exhibited evidence for maternally or paternally biased expression at multiple stages of endosperm development and are potential examples of differential imprinting. Our data indicate that parental effects on gene expression are much stronger in endosperm than in vegetative tissues.     

Structure and DNA methylation pattern of partially heterochromatinised endosperm nuclei in Gagea lutea (Liliaceae)

Pentaploid endosperm nuclei in certain Gagea species exhibit large masses of sticky and dense chromatin, not observed in somatic nuclei. These heterochromatin masses most probably stem from the triploid chalasal polar nucleus of the embryo sac, thus representing an example of facultative heterochromatinisation in plants. In the present investigation, we studied the nuclei in Gagea lutea (L.) Ker-Gawl. endosperm tissue. The position of the heterochromatin in interphase nuclei was observed by confocal laser scanning microscopy (CLSM) and the DNA methylation status of the euchromatin and heterochromatin was analysed by immunolabelling with an antibody raised against 5-methylcytosine (anti-5-mC). In young endosperms, heterochromatin was relatively dispersed, occupying some peripheral and inner parts of the nuclei. In a later endosperm development, the nuclei became smaller and more pycnotic, and the heterochromatin masses were placed predominantly near the nuclear periphery. The distribution of anti-5-mC labelling on the heterochromatic regions was unequal: some parts appeared hypermethylated while other parts were, like the euchromatin, not labelled. During mitosis, the labelling intensity of all the chromosomes was approximately the same, thus indicating that there are no cytologically detectable methylation differences among the individual sets of chromosomes. However, differences in the anti-5-mC signal intensity along individual chromosomes were observed, resulting in banding patterns with highly positive bands apparently representing constitutive heterochromatic regions. From these results it is obvious that facultative heterochromatinisation, in contrast to constitutive heterochromatinisation, need not be strictly accompanied by a prominent DNA hypermethylation. Received: 24 April 1997 / Accepted: 28 July 1997     

Gibberellinsäure-stimulierte Genaktivität im Endosperm von Phaseolus

Summary Gibberellic acid, injected into maturing ovules of Phaseolus vulgaris, induces 3.2-fold enhancement of the number of additional nucleoli within the endopolyploid endosperm nuclei. The additional nucleoli originate at different sites of polytene chromosome-like chromosome bundles. They contain RNA and are sensitive to actinomycin D. Similar nucleolar bodies are extruded by the main nucleolus. It is assumed that gibberellic acid stimulates the chromosomal and nucleolar RNA synthesis, i.e., the gene activity, in the endosperm nuclei.RNA synthesis was tested by ³H-thymidine it could be seen that gibberellin-treated and gibberellin-treated endosperms were labeled, but those of actinomycin-treated endosperms were not. Using ³H-thymidine it could be seen that gibberellin-treated endosperm nuclei continue to replicate their DNA for a longer period than untreated nuclei. Hence it follows that the treated nuclei become more highly endopolyploid and are capable of functioning longer than the untreated ones.

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Mit Unterstützung durch den Fonds zur Förderung der wissenschaftlichen Forschung der Republik Österreich.     

Hypomethylation promotes autonomous endosperm development and rescues postfertilization lethality in fie mutants

In most flowering plants, fertilization is necessary for development of the central cell into endosperm, but in the fie-1 mutant of Arabidopsis, the central cell can proliferate autonomously. However, autonomous fie-1 endosperms do not develop completely: They have fewer nuclei than sexually produced endosperms, cellularization does not take place, and no clear distinction is seen between the different endosperm compartments. Here, we show that autonomous endosperm develop much further in hypomethylated than normally methylated fie-1 mutants, undergoing cellularization and regional specification to resemble endosperm in sexually produced wild-type seeds. Therefore, the combination of maternal hypomethylation and loss of FIE function enables formation of differentiated endosperm without fertilization. A maternal fie-1 mutation is also lethal to sexual seeds, even if the pollen donor is wild type. We report that sexual mutant fie-1 endosperms fail to cellularize and overproliferate, consistent with the hypothesis that embryo abortion may be due, at least in part, to a defect in endosperm development. Finally, we show that pollen from hypomethylated plants rescues fie-1 mutant seeds provided that it also donates a wild-type paternal FIE allele. These results are discussed in light of models for parent-of-origin effects on seed development.     

基于F3种子的胚乳性状QTL区间定位

文章提出了包括胚乳效应和母体效应的胚乳性状QTL定位的统计方法,该方法的实验设计是分子标记基因型信息来自F2母体植株和F3种子胚(或植株),胚乳性状表型值来自F3单粒种子胚乳,称之为两步等级设计。同时,用计算机全面模拟以验证该模型的可行性,模拟结果表明,只要群体足够大,该模型能较有效地进行胚乳性状QTL定位并精确地估计出胚乳QTL的各种遗传效应和母体效应。     

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

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

The origin and maintenance of nuclear endosperms: viewing development through a phylogenetic lens

The endosperm develops in fertilized ovules of angiosperms following fertilization of the central cell and nuclei in the female gametophyte. Endosperms differ in whether, and which, nuclear divisions are followed by cellular divisions; the variants are classified as cellular, nuclear or helobial. Functional correlates of this variation are little understood. Phylogenetic methods provide a powerful means of exploring taxonomic variation and phylogenetic patterns, to frame questions regarding biological processes. Data on endosperms across angiosperms were analysed in a phylogenetic context in order to determine homologies and detect biases in the direction of evolutionary transitions. Analyses confirm that neither all nuclear nor all helobial endosperms are homologous, raise the possibility that cellular development is a reversal in some derived angiosperms (e.g. asterids) and show that a statistically significant bias towards evolution of nuclear endosperms (and against reversals) prevails in angiosperms as a whole. This bias suggests strong selective advantages to having nuclear endosperm, developmental constraints to reversals or both. Homologies suggest that the microtubular cycle and cellularization pattern characteristic of reproductive cells across land plants may have been independently co-opted during multiple origins of nuclear endosperms, but information on cellular endosperms is essential to investigate further.