AtMPK4 is required for male-specific meiotic cytokinesis in Arabidopsis

Mitogen-activated protein kinase (MAPK) cascades have been implicated in regulating various aspects of plant development, including somatic cytokinesis. The evolution of expanded plant MAPK gene families has enabled the diversification of potential MAPK cascades, but functionally overlapping components are also well documented. Here we report that Arabidopsis MPK4, an MAPK that was previously described as a regulator of disease resistance, can interact with and be phosphorylated by the cytokinesis-related MAP kinase kinase, AtMKK6. In mpk4 mutant plants, anthers can develop normal microspore mother cells (MMCs) and peripheral supporting tissues, but the MMCs fail to form a normal intersporal callose wall after male meiosis, and thus cannot complete meiotic cytokinesis. Nevertheless, the multinucleate mpk4 microspores subsequently proceed through mitotic cytokinesis, resulting in enlarged mature pollen grains that possess increased sets of the tricellular structure. This pollen development phenotype is reminiscent of those observed in both atnack2/tes/stud and anq1/mkk6 mutants, and protein-protein interaction analysis defines a putative signalling module linking AtNACK2/TES/STUD, AtANP3, AtMKK6 and AtMPK4 together as a cascade that facilitates male-specific meiotic cytokinesis in Arabidopsis.     

The DUET gene is necessary for chromosome organization and progression during male meiosis in Arabidopsis and encodes a PHD finger protein

Progression through the meiotic cell cycle is an essential part of the developmental program of sporogenesis in plants. The duet mutant of Arabidopsis was identified as a male sterile mutant that lacked pollen and underwent an aberrant male meiosis. Male meiocyte division resulted in the formation of two cells instead of a normal tetrad. In wild type, male meiosis extends across two successive bud positions in an inflorescence whereas in duet, meiotic stages covered three to five bud positions indicating defective progression. Normal microspores were absent in the mutant and the products of the aberrant meiosis were uni- to tri-nucleate cells that later degenerated, resulting in anthers containing largely empty locules. Defects in male meiotic chromosome organization were observed starting from diplotene and extending to subsequent stages of meiosis. There was an accumulation of meiotic structures at metaphase 1, suggesting an arrest in cell cycle progression. Double mutant analysis revealed interaction with dyad, a mutation causing chromosome cohesion during female meiosis. Cloning and molecular analysis of DUET indicated that it potentially encodes a PHD-finger protein and shows specific expression in male meiocytes. Taken together these data suggest that DUET is required for male meiotic chromosome organization and progression.     

Two novel meiotic restitution mechanisms in haploid maize (<Emphasis Type="Italic">Zea mays</Emphasis> 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.     

Correlated variation in microtubule distribution, callose deposition during male post-meiotic cytokinesis, and pollen aperture number across Nicotiana species (Solanaceae)

In most flowering plants, a single cytokinesis follows the two meiotic divisions during pollen-grain ontogeny. Aperture pattern (i.e., aperture number and distribution on pollen surface) ontogeny could be linked to the processes ensuring the apportionment of the cytoplasm to the four microspores.This apportionment is achieved by radial arrays of microtubules organized around the nuclei. The cleavage planes are defined in the overlapping regions of opposing arrays extending from different nuclei. We followed the establishment of these arrays in two different lines of plants belonging to the genus Nicotiana that produce pollen grains with different aperture numbers. Different distributions of the microtubules have been observed, which can be interpreted as resulting from variation in the interactions between nuclei; these distributions appear to be correlated with aperture number.As a consequence, we propose that simultaneous cytokinesis allows the formation of multiple pollen morphologies. This mechanism is consistent with aperture number distribution within angiosperms and provides clues to help our understanding of the evolution of aperture number.     

The microtubule‐associated kinase‐like protein RUNKEL functions in somatic and syncytial cytokinesis

The microtubule (MT)‐associated putative kinase RUNKEL (RUK) is an important component of the phragmoplast machinery involved in cell plate formation in Arabidopsis somatic cytokinesis. Since loss‐of‐function ruk mutants display seedling lethality, it was previously not known whether RUK functions in mature sporophytes or during gametophyte development. In this study we utilized RUK proteins that lack the N‐terminal kinase domain to further examine biological processes related to RUK function. Truncated RUK proteins when expressed in wild‐type Arabidopsis plants cause cellularization defects not only in seedlings and adult tissues but also during male meiocyte development, resulting in abnormal pollen and reduced fertility. Ultrastructural analysis of male tetrads revealed irregular and incomplete or absent intersporal cell walls, caused by disorganized radial MT arrays. Moreover, in ruk mutants endosperm cellularization defects were also caused by disorganized radial MT arrays. Intriguingly, in seedlings expressing truncated RUK proteins, the kinesin HINKEL, which is required for the activation of a mitogen‐activated protein kinase signaling pathway regulating phragmoplast expansion, was mislocalized. Together, these observations support a common role for RUK in both phragmoplast‐based cytokinesis in somatic cells and syncytial cytokinesis in reproductive cells.     

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.     

Analysis of gemini pollen 3 mutant suggests a broad function of AUGMIN in microtubule organization during sexual reproduction in Arabidopsis

In flowering plants, male gametes arise via meiosis of diploid pollen mother cells followed by two rounds of mitotic division. Haploid microspores undergo polar nuclear migration and asymmetric division at pollen mitosis I to segregate the male germline, followed by division of the germ cell to generate a pair of sperm cells. We previously reported two gemini pollen (gem) mutants that produced twin‐celled pollen arising from polarity and cytokinesis defects at pollen mitosis I in Arabidopsis. Here, we report an independent mutant, gem3, with a similar division phenotype and severe genetic transmission defects through pollen. Cytological analyses revealed that gem3 disrupts cell division during male meiosis, at pollen mitosis I and during female gametophyte development. We show that gem3 is a hypomorphic allele (aug6‐1) of AUGMIN subunit 6, encoding a conserved component in the augmin complex, which mediates microtubule (MT)‐dependent MT nucleation in acentrosomal cells. We show that MT arrays are disturbed in gem3/aug6‐1 during male meiosis and pollen mitosis I using fluorescent MT‐markers. Our results demonstrate a broad role for the augmin complex in MT organization during sexual reproduction, and highlight gem3/aug6‐1 mutants as a valuable tool for the investigation of augmin‐dependent MT nucleation and dynamics in plant cells.     

Meiotic mutants of Medicago sativa show altered levels of alpha- and beta-tubulin.

We have analysed the level of accumulation of alpha- and beta-tubulin polypeptides in flowers collected from different meiotic mutants of alfalfa (Medicago sativa L.). The H33 mutant previously identified as a producer of male and female gametes with the somatic chromosome number (2n gametes) as a result of defective spindle orientation or, more rarely, abnormal cytokinesis, showed a higher level of alpha- and beta-tubulin compared to control diploid plants and approximately the same level as control tetraploid plants. A higher level of tubulin was likewise observed in diploid plants displaying abnormalities in spindle orientation and cytokinesis, which had gone through 3-4 cycles of phenotypic recurrent selection to increase 2n gamete production. A similar analysis was performed on another class of Medicago meiotic mutants characterized by production of 4n pollen (jumbo pollen, due to the absence of cytokinesis at the end of meiosis) and 2n eggs. Again, the level of alpha- and beta-tubulin was found to be higher in the mutants than in diploid controls. We conclude that meiotic defects, such as abnormal spindle orientation or cytokinesis leading to the formation of 2n gametes, determine an increased level of tubulin, the main constituent of plant microtubules (MTs).     

A defect in synapsis causes male sterility in a T-DNA-tagged Arabidopsis thaliana mutant

Fluorescence microscopy was used to study meiosis in microsporocytes from wild-type Arabidopsis thaliana and a T-DNA-tagged meiotic mutant. Techniques for visualizing chromosomes and β-tubulin in other plant species were evaluated and modified in order to develop a method for analyzing meiosis in A. thaliana anthers. Like most dicots, A. thaliana microsporocytes undergo simultaneous cytokinesis in which both meiotic divisions are completed prior to cytokinesis. However, two unique events were observed in wild-type A. thaliana that have not been reported in other angiosperms: (1) polarization of the microsporocyte cytoskeleton during prophase I prior to nuclear envelope breakdown, and (2) extensive depolymerization of microtubules just prior to metaphase II. The first observation could have implications regarding a previously uncharacterized mechanism for determining the axis of the metaphase I spindle during microsporogenesis. The second observation is peculiar since microtubules are known to be involved in chromosome alignment in other species; possible explanations will be discussed. A T-DNA-tagged meiotic mutant of A. thaliana ( syn1 ), which had previously been shown to produce abnormal microspores with variable DNA content, was also cytologically characterized. The first observable defect occurs in microsporocytes at telophase I, where some chromosomes are scattered throughout the cytoplasm, usually attached to stray microtubules. Subsequent developmental stages are affected, leading to complete male sterility. Based on similarities to synaptic mutants that have been described in other species, it is suggested that this mutant is defective in synaptonemal complex formation and/or cohesion between sister chromatids.     

A Ploidy-Sensitive Mechanism Regulates Aperture Formation on the Arabidopsis Pollen Surface and Guides Localization of the Aperture Factor INP1

Pollen presents a powerful model for studying mechanisms of precise formation and deposition of extracellular structures. Deposition of the pollen wall exine leads to the generation of species-specific patterns on pollen surface. In most species, exine does not develop uniformly across the pollen surface, resulting in the formation of apertures–openings in the exine that are species-specific in number, morphology and location. A long time ago, it was proposed that number and positions of apertures might be determined by the geometry of tetrads of microspores–the precursors of pollen grains arising via meiotic cytokinesis, and by the number of last-contact points between sister microspores. We have tested this model by characterizing Arabidopsis mutants with ectopic apertures and/or abnormal geometry of meiotic products. Here we demonstrate that contact points per se do not act as aperture number determinants and that a correct geometric conformation of a tetrad is neither necessary nor sufficient to generate a correct number of apertures. A mechanism sensitive to pollen ploidy, however, is very important for aperture number and positions and for guiding the aperture factor INP1 to future aperture sites. In the mutants with ectopic apertures, the number and positions of INP1 localization sites change depending on ploidy or ploidy-related cell size and not on INP1 levels, suggesting that sites for aperture formation are specified before INP1 is brought to them.     

RUPTURED POLLEN GRAIN1, a member of the MtN3/saliva gene family, is crucial for exine pattern formation and cell integrity of microspores in Arabidopsis

During microsporogenesis, the microsporocyte (or microspore) plasma membrane plays multiple roles in pollen wall development, including callose secretion, primexine deposition, and exine pattern determination. However, plasma membrane proteins that participate in these processes are still not well known. Here, we report that a new gene, RUPTURED POLLEN GRAIN1 (RPG1), encodes a plasma membrane protein and is required for exine pattern formation of microspores in Arabidopsis (Arabidopsis thaliana). The rpg1 mutant exhibits severely reduced male fertility with an otherwise normal phenotype, which is largely due to the postmeiotic abortion of microspores. Scanning electron microscopy examination showed that exine pattern formation in the mutant is impaired, as sporopollenin is randomly deposited on the pollen surface. Transmission electron microscopy examination further revealed that the primexine formation of mutant microspores is aberrant at the tetrad stage, which leads to defective sporopollenin deposition on microspores and the locule wall. In addition, microspore rupture and cytoplasmic leakage were evident in the rpg1 mutant, which indicates impaired cell integrity of the mutant microspores. RPG1 encodes an MtN3/saliva family protein that is integral to the plasma membrane. In situ hybridization analysis revealed that RPG1 is strongly expressed in microsporocyte (or microspores) and tapetum during male meiosis. The possible role of RPG1 in microsporogenesis is discussed.     

Arabidopsis Fused kinase TWO-IN-ONE dominantly inhibits male meiotic cytokinesis

Arabidopsis Fused kinase TWO-IN-ONE (TIO) controls phragmoplast expansion through its interaction with the Kinesin-12 subfamily proteins that anchor the plus ends of interdigitating microtubules in the phragmoplast midzone. Previous analyses of loss-of-function mutants and RNA interference lines revealed that TIO positively controls both somatic and gametophytic cell cytokinesis; however, knowledge of the full spectrum of TIO functions during plant development remains incomplete. To characterize TIO functions further, we expressed TIO and a range of TIO variants under control of the TIO promoter in wild-type Arabidopsis plants. We discovered that TIO-overexpressing transgenic lines produce enlarged pollen grains, arising from incomplete cytokinesis during male meiosis, and show sporophytic abnormalities indicative of polyploidy. These phenotypes arose independently in TIO variants in which either gametophytic function or the ability of TIO to interact with Kinesin-12 subfamily proteins was abolished. Interaction assays in yeast showed TIO to bind to the AtNACK2/TETRASPORE, and plants doubly homozygous for kinesin-12a and kinesin-12b knockout mutations to produce enlarged pollen grains. Our results show TIO to dominantly inhibit male meiotic cytokinesis in a dosage-dependent manner that may involve direct binding to a component of the canonical NACK-PQR cytokinesis signaling pathway.     

LP28, a lily pollen-specific LEA-like protein, is located in the callosic cell wall during male gametogenesis

. LP28, a pollen-specific LEA-like protein identified in Lilium longiflorum purportedly related to the desiccation tolerance of pollen, was localized during male gametogenesis using immuno-electron microscopy. At premeiotic interphase, LP28 label is absent from the microsporocyte. LP28 label was first detected in the cell wall of the microsporocyte at meiotic prophase I. LP28 gradually increased as the cell wall thickened. In the dyad, after the first meiotic division, LP28 label also appeared in the septum. In the tetrad, after the second meiotic division, LP28 was detected throughout the cell wall, including the septa. Immunolabeling of callose during meiosis indicated that the appearance and localization of LP28 was very similar to that of callose. After the microspores were released from the tetrad by digesting the callosic cell wall, LP28 was not found in the microspores. In bicellular pollen, just after microspore mitosis, LP28 appeared in the generative cell wall, which also consisted of callose. After pollen germination, LP28 also accumulated in the callosic layer of the elongated pollen tube wall and the callose plug. Thus, LP28 colocalized with the callosic cell wall during male gametogenesis. The possible role of LP28 with respect to wall formation during meiosis and pollen development is discussed.     

矮牡丹小孢子发生和雄配子体发育及其与该种濒危的关系

研究了矮牡丹Paeonia jishanensis Hong et W．Z．Zhao的小孢子发生及雄配子体的形成。矮 牡丹花药具4个小孢子囊,药壁结构属双子叶型,腺质绒毡层,小孢子母细胞减数分裂后胞质分裂为 同时型,四分体多为四面体形,少左右对称形,成熟花粉为2-细胞。对芍药属木本类型的雄性发育进行 了全面研究,还对小孢子母细胞减数分裂和单核小孢子发育时期的异常现象进行了观察,对能育花粉 与不育花粉的百分比进行了测定,结果表明,能育花粉为45.03％～84.18％,它们在不同花中,不同花 药中,甚至同一花药的不同花粉囊中表现都不完全一致。联系矮牡丹的致濒原因进行了讨论,认为雄配子体形成过程中的异常现象,并不是导致矮牡丹濒危的主要因素。     

The Arabidopsis Gene Tardy Asynchronous Meiosis Is Required for the Normal Pace and Synchrony of Cell Division during Male Meiosis

Male meiosis in higher organisms features synchronous cell divisions in a large number of cells. It is not clear how this synchrony is achieved, nor is it known whether the synchrony is linked to the regulation of cell cycle progression. Here, we describe an Arabidopsis mutant, named tardy asynchronous meiosis (tam), that exhibits a phenotype of delayed and asynchronous cell divisions during male meiosis. In Arabidopsis, two nuclear divisions occur before simultaneous cytokinesis yields a tetrad of haploid cells. In tam, cell divisions are delayed, resulting in the formation of abnormal intermediates, most frequently dyad meiotic products, or in rare cases, dyad pollen (two gametophytes within one exine wall). Temperature-shift experiments showed that the percentage of the abnormal intermediates increased at 27 degrees C. Analysis of tam and the tam/quartet1 double mutant showed that most of these abnormal intermediates could continue through the normal rounds of cell divisions and form functional pollen, though at a slower than normal pace. The asynchrony of cell division started at the G2/M transition, with cells entering metaphase at different time points, during both meiosis I and II. In addition, chromosome condensation defects and mis-segregation were sometimes observed in tam. These observations suggest that the TAM protein positively regulates cell cycle progression, perhaps by promoting the G2/M transition. We speculate that there is a signal, perhaps TAM, that couples the normal pace of cell cycle progression with the synchrony of cell division during male meiosis.     

Minispindles and cytoplasmic domains in microsporogenesis of orchids

Summary Changes in the microtubular cytoskeleton during meiosis and cytokinesis in hybrid moth orchids were studied by indirect immunofluorescence. Lagging chromosomes not incorporated into telophase nuclei after first meiotic division behave as small extra nuclei. Events in the microtubular cycle associated with these micronuclei are similar to and synchronous with those of the principal nuclei. During second meiotic division the micronuclei trigger formation of minispindles which are variously oriented with respect to the two principal spindles. After meiosis, radial systems of microtubules measure cytoplasmic domains around each nucleus in the coenocyte. Cleavage planes are established in regions where opposing radial arrays interact and the cytoplasm cleaved around micronuclei is proportionately smaller than that around the four principal nuclei. These observations clearly demonstrate that nuclei in plant cells are of fundamental importance in microtubule organization and provide strong evidence in support of our recently advanced hypothesis that division planes in simultaneous cytokinesis following meiosis are determined by establishment of cytoplasmic domains via radial systems of nuclear-based microtubules rather than by division sites established before nuclear division.Abbreviations DMSO dimethylsulfoxide - FITC fluorescein isothiocyanate - MTOC microtubule organizing center - PBS phosphate buffered saline - PPB preprophase band of microtubules     

The Arabidopsis MEI1 gene encodes a protein with five BRCT domains that is involved in meiosis-specific DNA repair events independent of SPO11-induced DSBs

Arabidopsis thaliana MEI1 was first described as a gene involved in male meiosis, encoding a short protein showing homology with a human acrosin-trypsin inhibitor. We have isolated a new allele of mei1, and shown that in both mutants male and female meiosis are affected. In both reproductive pathways, meiosis proceeds while chromosomes become fragmented, resulting in aberrant meiotic products and in a strongly reduced fertility. We have shown that the gene mutated in mei1 mutants actually encodes a protein of 972 amino acids that contains five BRCA1 C-terminus (BRCT) domains and is similar to proteins involved in the response to DNA damage and replication blocks in eukaryotes. During meiosis, recombination is initiated by the formation of DNA double strand breaks (DSBs) induced by the protein SPO11. We analysed meiotic chromosome behaviour of the mei1 mutant in a spo11 mutant background and proved that the meiotic fragmentation observed in mei1 mutants was not the consequence of defects in the repair of meiotic DSBs induced by SPO11. We also analysed the effect of mei1 on the mitotic cell cycle but could not detect any sensitivity of mei1 seedlings to DNA-damaging agents like gamma-rays or UV. Therefore, MEI1 is a BRCT-domain-containing protein that could be specific to the meiotic cell cycle and that plays a crucial role in some DNA repair events independent of SPO11 DSB recombination repair.     

Absence of microspore polarity, symmetric divisions and pollen cell fate in Brachiaria decumbens (Gramineae).

Meiotic division and male gametophyte development were analyzed in one tetraploid (2n = 4x = 36) accession of Brachiaria decumbens cv. Basilisk that showed some pollen sterility. Meiotic process was typical of polyploids in that it consisted of multiple chromosome associations. Precocious chromosome migration to the poles, laggards, and micronucleus formation were abundant in both meiosis I and II and resulted in tetrads with micronuclei. After callose dissolution, microspores were released into the anther locule and had the semblance of being normal. Although each microspore initiated its differentiation by pollen mitosis, in 43.24% of the microspores, nuclear polarization was not observed and the typical hemispherical cell plate was not detected. Division was symmetric and microspores lacked differentiation between the vegetative and the generative cell. Both nuclei were of equal size, presented equal chromatin condensation, and had a spherical shape. After the first pollen mitosis and cytokinesis, each cell underwent a new symmetric mitosis without nuclear polarization. At the end of the second pollen mitosis, four equal nuclei were observed in each pollen grain. After the second cytokinesis, the cells gave rise to four equal-sized pollen grains with a similar tetrad configuration that initially remained together. Sterile pollen grains resulted from abnormal pollen mitosis. This anomaly may be explained by a mutation, probably affecting microtubule cytoskeleton formation. The importance of this male-sterile mutation for Brachiaria breeding programs is discussed.