Two novel meiotic restitution mechanisms in haploid maize (Zea mays L.)

Two original mechanisms of nuclear restitution related to different processes of meiotic division of pollen mother cells (PMCs) have been found in male meiosis of the lines of maize haploids no. 2903 and no. 2904. The first mechanism, which is characteristic of haploid no. 2903, consists in spindle deformation (bend) in the conventional metaphase-anaphase I. This leads to asymmetric incomplete cytokinesis with daughter cell membranes in the form of incisions on the mother cell membrane. As a result, the chromosomes of the daughter nuclei are combined into a common spindle during the second meiotic division, and a dyad of haploid microspores is formed at the tetrad stage. The frequency of this abnormality is about 50%. The second restitution mechanism, which has been observed in PMCs of haploid no. 2904, results from disturbance of the fusion of membrane vesicles (plastosomes) at the moment of formation of daughter cell membranes and completion of cytokinesis in the first meiotic division. This type of cell division yields a binuclear monad. In the second meiotic division, the chromosomes of the daughter nuclei form a common spindle, and meiosis results in a dyad of haploid microspores. The frequency of this abnormality is as high as 15%. As a result, haploid lines no. 2903 and no. 2904 partly restore fertility.     

Cortical cytoskeletal ring in prophase II leads to correction of abnormalities of the first meiotic division and to meiotic restitution of pollen mother cell nucleus

The deviation of prophase cytoskeletal ring formation was determined during meiotic division in 50% of pollen mother cells (PMCs) in maize haploid No 1498 (Zea mays). At prophase in both meiotic divisions the cytoskeletal ring is formed in cortical region of cytoplasm instead of perinuclear. Sometimes formation of both perinuclear and cortical rings is observed in the same cell. It has been shown that in multinucleate PMCs the cortical ring leads to the consolidation of chromosomes into common spindle and to meiotic restitution.     

Cytokinesis in plant male meiosis

In somatic cell division, cytokinesis is the final step of the cell cycle and physically divides the mother cytoplasm into two daughter cells. In the meiotic cell division, however, pollen mother cells (PMCs) undergo two successive nuclear divisions without an intervening S-phase and consequently generate four haploid daughter nuclei out of one parental cell. In line with this, the physical separation of meiotic nuclei does not follow the conventional cytokinesis pathway, but instead is mediated by alternative processes, including polar-based phragmoplast outgrowth and RMA-mediated cell wall positioning. In this review, we outline the different cytological mechanisms of cell plate formation operating in different types of PMCs and additionally focus on some important features associated with male meiotic cytokinesis, including cytoskeletal dynamics and callose deposition. We also provide an up-to-date overview of the main molecular actors involved in PMC wall formation and additionally highlight some recent advances on the effect of cold stress on meiotic cytokinesis in plants.     

“Bouquet arrest”, monopolar chromosomes segregation, and correction of the abnormal spindle

According to our data, the arrest of univalents in bouquet arrangement is a widespread meiotic feature in cereal haploids and allohaploids (wide hybrids F₁). We have analyzed 83 different genotypes of cereal haploids and allohaploids with visualization of the cytoskeleton and found a bouquet arrest in 45 of them (in 30% to 100% pollen mother cells (PMCs)). The meiotic plant cell division in 26 various genotypes with a zygotene bouquet arrest was analyzed in detail. In three of them in PMCs, a very specific monopolar conic-shaped figure at early prometaphase is formed. This monopolar figure consists of mono-oriented univalents and their kinetochore fibers converging in pointed pole. Such figures are never observed at wild-type prometaphase or in asynaptic meiosis in the variants without a bouquet arrest. Later at prometaphase, the bipolar central spindle fibers join in this monopolar figure, and a bipolar spindle with all univalents connected to one pole is formed. As a result of monopolar chromosome segregation at anaphase and normal cytokinesis at telophase, a dyad with one member carrying a restitution nucleus and the other enucleated is formed. However, such phenotype has only three genotypes among 26 analyzed with a bouquet arrest. In the remaining 23 haploids and allohaploids, the course of prometaphase was altered after the conic monopolar figure formation. In these variants, the completely formed conic monopolar figure was disintegrated into a chaotic network of spindle fibers and univalents acquired a random orientation. This arrangement looks like a mid-prometaphase in the wild-type meiosis. At late prometaphase, a bipolar spindle is formed with the univalents distributed more or less equally between two poles, similar to the phenotypes without a bouquet arrest. The product of cell division is a dyad with aneuploid members. Thus, the spindle abnormality—monopolar chromosome orientation—is corrected. In some cells the correction of the prometaphase monopolus occurs by means of its splitting into two half-spindles and their rotation along the future division axis.     

Extreme cytokinesis phenomenon in the phenotype of meiotic mutation pam of Zea mays

Our study of cytological phenotype of meiotic mutation pam resulted in detecting a failure of cytokinesis in mutant pollen mother cells in the form of a block of fusion of membrane vesicles of the cell plate, and an impossibility of formation of daughter cell membranes. The mutation does not disturb the division spindle structure and function. Asynchrony of meiosis in pam is the result of arrest of pollen mother cells at metaphase 1 and metaphase 2.     

Premature cytokinesis in pollen mother cells of transgenic tobacco plants Nicotiana tabacum L

In majority species of dicotyledonous plants cytokinesis in PMCs occurs once after completion of two caryokinesis cycles, that is a simultaneous type. This paper represents cytological picture and frequency characteristics of abnormality which resulted in cytokinesis triggering after first meiotic division in a part of transgenic tobacco PMCs. It was shown that the main process of cytoskeleton reorganization typical for simultaneous cytokinesis remained without any alterations in such cells. However, in most cases premature cell division occurred with abnormalities such as membrane "tunnel" or "gash" formation. It was ascertained that initiation of additional round ofcytokinesis did not block nuclear cycle and cytokinesis after second meiotic division. Thus, transition of cell division from simultaneous type to successive one occurs under this abnormality.     

Microtubule distribution in dv, a maize meiotic mutant defective in the prophase to metaphase transition

Microsporogenesis in Zea mays, the meiotic reduction of diploid sporocytes to haploid microspores, proceeds through a well-defined developmental sequence. The ability to generate mutants that affect the process makes this an ideal system for elucidating the role of the cytoskeleton during plant development. We have used immunofluorescence microscopy to compare microtubule distribution in wild-type and mutant microsporocytes. During normal meiosis the distribution of microtubules follows a specific temporal and spatial pattern that reflects the polar nature of microspore formation. Perinuclear microtubule staining increases and the nucleus elongates in the future spindle axis during late prophase I. Metaphase I spindles with highly focused poles align along the long axis of the anther locule. Cytokinesis occurs perpendicular to the spindle axis. The second division axis shifts 90 degrees with respect to the first division plane, thereby yielding an isobilateral tetrad of microspores. Microtubule distribution patterns during meiosis suggest that a nuclear envelope-associated microtubule organizing center (MTOC) controls the organization of cytoplasmic microtubules and contributes to spindle formation. The meiotic mutant dv is defective in the transition from a prophase microtubule array to a metaphase spindle. Instead of converging to form focused poles, the metaphase spindle poles remain diffuse as in prometaphase. This defect correlates with several abnormalities in subsequent developmental events including the formation of multinucleate daughter cells, multiple microspindles during meiosis II, multiple phragmoplasts, polyads of microspores, and cytoplasmic microtubule foci. These results suggest that dv is a mutation that affects MTOC organization.     

Evolution of meiotic patterns of oogenesis and spermatogenesis in centric diatoms

The evolution of meiotic patterns of the oogenesis and spermatogenesis in centric diatoms was inferred according to the parsimony principle. The pattern provisionally named type 1, in which one of two daughter nuclei becomes pycnotic, no cytokinesis occurs at meiosis II and two eggs are produced, was inferred to be the most primitive among extant meiotic patterns of oogenesis. It was also inferred that the pattern provisionally named 4‐2EC, in which equal cytokinesis occurs after each nuclear division and four functional haploid cells are produced, is the most primitive among extant meiotic patterns of spermatogenesis. The evolution of meiotic pattern suggests that bipolar or multipolar forms are primitive among centric diatoms.     

Cytological evidences of SDR-FDR mixture in the formation of 2n eggs in a potato diploid clone

Summary Macrosporogenesis and microsporogenesis were investigated in a diploid S. tuberosum x S. chacoense potato hybrid, characterized by more than 50% 2n egg formation. Fifty-five percent of dyad formation of 2n macrospores is ascribed to two meiotic abnormalities: omission of the second meiotic division, occurring at a frequency of 38%, and irregular spindle axis orientation at metaphase I at a frequency of 16%: These abnormalities give origin to a mixture of 2n eggs, composed of mostly second division restitution (SDR) and a small portion of first division restitution (FDR). Microsporogenesis showed rare dyads of 2n microspores depending on parallel spindles observed in anaphase II.Contribution no. 53 from the Center of Vegetable Breeding, CNR, Portici, Italy     

{gamma}-Tubulin and microtubule organization during meiosis in the liverwort Ricciocarpus natans (Ricciaceae)

Extant liverworts are "living fossils" considered sister to all other plants and as such provide clues to the evolution of the microtubule organizing center (MTOC) in anastral cells. This report is the first on microtubule arrays and their γ-tubulin-nucleating sites during meiosis in a member of the Ricciales, a specialized, species-rich group of complex thalloid (marchantioid) liverworts. In meiotic prophase, γ-tubulin becomes concentrated at several sites adjacent to the nuclear envelope. Microtubules organized at these foci give rise to a multipolar prometaphase spindle. By metaphase I, the spindle has matured into a bipolar structure with truncated poles. In both first and second meiosis, γ-tubulin forms box-like caps at the spindle poles. γ-Tubulin moves from spindle poles to the proximal surfaces of telophase chromosomes where interzonal microtubules are nucleated. Although a phragmoplast is organized, no cell plate is deposited, and second division occurs simultaneously in the undivided sporocyte. γ-Tubulin surrounds each of the tetrad nuclei, and phragmoplasts initiated between both sister and nonsister nuclei direct simultaneous cytokinesis. The overall pattern of meiosis (unlobed polyplastidic sporocytes, nuclear envelope MTOC, multipolar spindle origin, spindles with box-like poles, and simultaneous cytokinesis) more closely resembles that of Conocephalum than other marchantiod liverworts.     

Cytological studies on meiosis and male gametophyte development in autotetraploid cucumber

With improved staining and chromosome preparation techniques, meiosis of pollen mother cells (PMCs) and male gametophyte development in autotetraploid cucumber (Cucumis sativus L.) was studied to understand the correlation between chromosomes behaviour and fertility. Various chromosome configurations, e.g. multivalent, quadrivalents, trivalents, bivalents and univalents were observed in most PMCs at metaphase I. Lagging chromosomes were frequently observed at anaphase in both meiotic divisions. In addition, chromosomes segregations were not synchronous and equal in some PMCs during anaphase II and telophase II. Dyads, triads, tetrads with micronuclei and polyads were observed at tetrad stage, and the frequencies of normal tetrad with four microcytes were only 55.4 %. The frequency of abnormal behaviour in each stage of meiosis was counted, and the average value was 37.2 %. The normal meiotic process could be accomplished to form the microspore tetrads via simultaneous cytokinesis. Most microspores could develop into fertile gametophytes with 2 cells and 3 germ pores through the following stages: single-nucleus early stage, single-nucleus late stage and 2-celled stage. The frequency of abnormalities was low during the process of male gametophyte development. The germination rate of pollen grains was 46.9 %. These results suggested that abnormal meiosis in PMCs was the reason for low pollen fertility in the autotetraploid cucumber.     

Centriolin,a centriole-appendage protein,regulates peripheral spindle migration and asymmetric division in mouse meiotic oocytes

Unlike somatic cells mitosis, germ cell meiosis consists of 2 consecutive rounds of division that segregate homologous chromosomes and sister chromatids, respectively. The meiotic oocyte is characterized by an absence of centrioles and asymmetric division. Centriolin is a relatively novel centriolar protein that functions in mitotic cell cycle progression and cytokinesis. Here, we explored the function of centriolin in meiosis and showed that it is localized to meiotic spindles and concentrated at the spindle poles and midbody during oocyte meiotic maturation. Unexpectedly, knockdown of centriolin in oocytes with either siRNA or Morpholino micro-injection, did not affect meiotic spindle organization, cell cycle progression, or cytokinesis (as indicated by polar body emission), but led to a failure of peripheral meiotic spindle migration, large polar body emission, and 2-cell like oocytes. These data suggest that, unlike in mitotic cells, the centriolar protein centriolin does not regulate cytokinesis, but plays an important role in regulating asymmetric division of meiotic oocytes.     

Mechanisms that prevent catastrophic interactions between paternal chromosomes and the oocyte meiotic spindle

Meiosis produces haploid gametes by accurately reducing chromosome ploidy through one round of DNA replication and two subsequent rounds of chromosome segregation and cell division. The cell divisions of female meiosis are highly asymmetric and give rise to a large egg and two very small polar bodies that do not contribute to development. These asymmetric divisions are driven by meiotic spindles that are small relative to the size of the egg and have one pole juxtaposed against the cell cortex to promote polar body extrusion. An additional unique feature of female meiosis is that fertilization occurs before extrusion of the second polar body in nearly all animal species. Thus sperm-derived chromosomes are present in the egg during female meiosis. Here, we explore the idea that the asymmetry of female meiosis spatially separates the sperm from the meiotic spindle to prevent detrimental interactions between the spindle and the paternal chromosomes.     

Meiotic division and fusion of nuclei in multinucleate cells from the induced fusion of meiotic protoplasts of liliaceous plants

Multinucleate protoplasts were produced from meiotic cells at the zygotene and pachytene stages in a lily andTrillium, and their meiotic divisions were followed during subsequent culture. In each multinucleate, a complete synchrony of nuclear division was maintained throughout the meiotic process, and chromosome behavior appeared normal up to the metaphase stage. In most dinucleates, chromosome segregation movement was organized in a common spindle, and the daughter nuclei at the telophase appeared to envelope each other in the newly formed nuclear membrane. The cell was divided into two daughter cells by a common cell plate. Trinucleates were similarly converted to two cells with a hexaploid number of chromosomes. Some of the di- and trinucleates subsequently completed the second meiotic division with the formation of typical tetrad configurations. In giant cells with more than several nuclei, chromosomes separated at random but reaggregated into one giant resting nucleus, with no later cytokinesis. The rate of meiotic development in multinucleates was relatively slower in cells which contained greater numbers of nuclei.     

The spatial and mechanical challenges of female meiosis

Recent work shows that cytokinesis and other cellular morphogenesis events are tuned by an interplay among biochemical signals, cell shape, and cellular mechanics. In cytokinesis, this includes cross-talk between the cortical cytoskeleton and the mitotic spindle in coordination with cell cycle control, resulting in characteristic changes in cellular morphology and mechanics through metaphase and cytokinesis. The changes in cellular mechanics affect not just overall cell shape, but also mitotic spindle morphology and function. This review will address how these principles apply to oocytes undergoing the asymmetric cell divisions of meiosis I and II. The biochemical signals that regulate cell cycle timing during meiotic maturation and egg activation are crucial for temporal control of meiosis. Spatial control of the meiotic divisions is also important, ensuring that the chromosomes are segregated evenly and that meiotic division is clearly asymmetric, yielding two daughter cells - oocyte and polar body - with enormous volume differences. In contrast to mitotic cells, the oocyte does not undergo overt changes in cell shape with its progression through meiosis, but instead maintains a relatively round morphology with the exception of very localized changes at the time of polar body emission. Placement of the metaphase-I and -II spindles at the oocyte periphery is clearly important for normal polar body emission, although this is likely not the only control element. Here, consideration is given to how cellular mechanics could contribute to successful mammalian female meiosis, ultimately affecting egg quality and competence to form a healthy embryo.     

Microtubule organization during successive microsporogenesis in Allium cepa and simultaneous cytokinesis in Nicotiana tabacum

Microtubule cytoskeleton organization during microspore mother cell (MMC) meiosis in Allium cepa L. and microsporogenesis in Nicotiana tabacum L. was examined. The MMC microtubules (MTs) were short and well dispersed in the cytoplasm of both taxa. As the MMCs of both species entered metaphase of meiosis I, the MTs constructed a spindle that facilitated the chromosomes to orient in the meridian plane. At anaphase of meiosis I, the spindle MTs differentiated into two types: one MT type became short, pulled the chromosomes toward the two poles, and was designated as centromere MTs; the second type of MT connected the two poles, and was designated as pole MTs. In A. cepa, where successive cytokinesis was observed, pole MTs assumed a tubbish shape. Some new short MTs aggregated in the meridian plane and constricted to form a phragmoplast, which developed into a cell plate, divided the cytoplasm into two parts and produced a dyad. However, in tobacco, a phragmoplast was not generated in anaphase of meiosis I and II and cytokinesis did not occur. The spindle MTs depolymerized and reorganized the radial arrangement of MTs from the nucleate surface to the periplasm during anaphase. Following telophase of meiosis II, the cytoplasm produced centripetal furrows, which met in the center of the cell and divided it into four parts, serving as a form of cytokinesis. In this process, MTs appeared to bear no relationship to cytokinesis.     

Meiotic restitution in amphihaploids in the tribe Triticeae

In haploid and diploid organisms of the plant kingdom, meiotic division of diploid cells proceeds in two consecutive stages, with DNA replicating only once. In amphihaploids (interspecific or intergeneric hybrids), where homologs are absent, the reduction of the chromosome number does not occur, meiosis is abnormal, and the plants are sterile. Gamete viability in F₁ hybrids is ensured by a single division when chromosomes are separated into sister chromatids in either the first or the second division. Such gametes ensure partial fertility of amphihaploids, thereby facilitating their survival and stabilization of the polygenome. The frequency of the formation of viable gametes varies from a few cases to 98.8% in different anthers of the hybrids. Here, studies on the cytological mechanisms and genetic control of chromosome unreduction or restitution in different amphihaploids of the tribe Triticeae are reviewed. The current notions on the control of formation of restitution nuclei based on the principles of a prolonged metaphase I and different types of meiocytes. The main terms used for systematization of restitution mechanisms are first-division restitution (FDR), single-division meiosis (SDM), and unreductional meiotic cell division (UMCD). It has been assumed that archesporial cells of wide hybrids may have two cell division programs, the meiotic and the mitoyic ones The possible approaches to the analysis of the genetic control of chromosome restitution in amphihaploids are discussed.     

Division polarity and plasticity of the meiosis I spindle inCypripedium californicum (Orchidaceae)

Summary Establishment of division polarity and meiotic spindle organization in the lady's slipper orchidCypripedium californicum A. Gray was studied by immunocytochemistry, confocal and transmission electron microscopy. Prior to organization of the spindle for meiosis I, the cytoplasmic domains of the future dyad and spindle polarity are marked by: (1) constriction of the prophase nucleus into an hourglass shape; (2) reorganization of nuclear-based radial microtubules into two arrays that intersect at the constriction; and (3) redistribution of organelles into a ring at the boundary of the newly defined dyad domains. It is not certain whether the opposing microtubule arrays contribute directly to the anastral spindle which is organized in the perinuclear areas of the two hemispheres. By late prophase each half-spindle consists of a spline-like structure from which depart the kinetochore fibers. This peculiar spindle closely resembles the spline-like spindle of generative-cell mitosis in certain plants where the spindle is distorted by physical constraints of the slender pollen tube. In the microsporocyte, the elongate spindle of late prophase/metaphase is curved within the cell so that the poles are not actually opposite each other and chromosomes do not form a plate at the equator. By late telophase the poles of the shortened halfspindles lie opposite each other. Plasticity of the physically constrained plant spindle appears to be due to its construction from multiple units terminating in minipoles. Cytokinesis does not follow the first meiosis. However, the dyad domains are clearly defined by radial microtubules emanating from the two daughter nuclei and the domains themselves are separated by a disc-like band of organelles.     

The dyad gene is required for progression through female meiosis in Arabidopsis

In higher plants the gametophyte consists of a gamete in association with a small number of haploid cells, specialized for sexual reproduction. The female gametophyte or embryo sac, is contained within the ovule and develops from a single cell, the megaspore which is formed by meiosis of the megaspore mother cell. The dyad mutant of Arabidopsis, described herein, represents a novel class among female sterile mutants in plants. dyad ovules contain two large cells in place of an embryo sac. The two cells represent the products of a single division of the megaspore mother cell followed by an arrest in further development of the megaspore. We addressed the question of whether the division of the megaspore mother cell in the mutant was meiotic or mitotic by examining the expression of two markers that are normally expressed in the megaspore mother cell during meiosis. Our observations indicate that in dyad, the megaspore mother cell enters but fails to complete meiosis, arresting at the end of meiosis 1 in the majority of ovules. This was corroborated by a direct observation of chromosome segregation during division of the megaspore mother cell, showing that the division is a reductional and not an equational one. In a minority of dyad ovules, the megaspore mother cell does not divide. Pollen development and male fertility in the mutant is normal, as is the rest of the ovule that surrounds the female gametophyte. The embryo sac is also shown to have an influence on the nucellus in wild type. The dyad mutation therefore specifically affects a function that is required in the female germ cell precursor for meiosis. The identification and analysis of mutants specifically affecting female meiosis is an initial step in understanding the molecular mechanisms underlying early events in the pathway of female reproductive development.     

Nuclear cytoplasmic domains,microtubules and organelles in microsporocytes of the slipper orchid Cypripedium californicum A. Gray dividing by simultaneous cytokinesis

Microsporocytes of the slipper orchidCypripedium californicum A. Gray divide simultaneously after second meiosis. The organization and apportionment of the cytoplasm throughout meiosis are functions of nuclear-based radial microtubule systems (RMSs) that define domains of cytoplasm - a single sporocyte domain before meiosis, dyad domains within the undivided cytoplasm after first meiosis, and four spore domains after second meiosis. Organelles migrate to the interface of dyad domains in the undivided cytoplasm after first meiotic division, and second meiotic division takes place simultaneously on both sides of the equatorial organelle band. Microtubules emanating from the telophase II nuclei interact to form columnar arrrays that interconnect all four nuclei, non-sister as well as sister. Cell plates are initiated in these columns of microtubules and expand centrifugally along the interface of opposing RMSs, coalescing in the center of the sporocyte and joining with the original sporocyte wall at the periphery to form the tetrad of microspores. Organelles are distributed into the spore domains in conjunction with RMSs. These data, demonstrating that cytokinesis in microsporogenesis can occur in the absence of both components of the typical cytokinetic apparatus (the preprophase band of microtubules which predicts the division site and the phragmoplast which controls cell-plate deposition), suggest that plant nuclei have an inherent ability to establish a domain of cytoplasm via radial microtubule systems and to regulate wall deposition independently of the more complex cytokinetic apparatus of vegetative cells.