Evolution of Parallel Spindles Like genes in plants and highlight of unique domain architecture#

Background  

Polyploidy has long been recognized as playing an important role in plant evolution. In flowering plants, the major route of polyploidization is suggested to be sexual through gametes with somatic chromosome number (2n). Parallel Spindle1 gene in Arabidopsis thaliana (AtPS1) was recently demonstrated to control spindle orientation in the 2nd division of meiosis and, when mutated, to induce 2n pollen. Interestingly, AtPS1 encodes a protein with a FHA domain and PINc domain putatively involved in RNA decay (i.e. Nonsense Mediated mRNA Decay). In potato, 2n pollen depending on parallel spindles was described long time ago but the responsible gene has never been isolated. The knowledge derived from AtPS1 as well as the availability of genome sequences makes it possible to isolate potato PSLike (PSL) and to highlight the evolution of PSL family in plants.     

Meiosis spindle formation and chromosome behavior in diploid potatoes with 'fused spindles' mutation

Chromosomal behaviour and spindle morphology were studied in microsporogenesis of two kinds of diploid potato clones: with normal meiosis, and with "fused spindles" (fs) occurring during the second meiotic division from prometaphase II (proMII) to telophase II (TII). For the first time, morphological effect of fs was found at the late proMII stage to be expressed as two interrelated processes: 1) abnormal chromosome movement, which resulted in joining two groups of chromosomes in the central zone of meiocytes, and 2) abnormal formation of two spindles in the direction to two division poles instead of four poles that actually led to the formation of a united bipolar spindle. Thus, it is not the fusion of two parallel spindles but the formation of united bipolar spindle that constitutes fs abnormality, while the parallel co-orientation of two spatially separated meiotic spindles is a norm in diploid potato. These primary abnormalities detected at proMII resulted in abnormalities at its subsequent meiotic stages: formation of fused spindle and united metaphase plate at MII, bipolar chromosome segration at anaphase II, formation of two telophase nuclei at TII and dyads at the tetrad stage. The results obtained evidence the polar division disturbance in diploid potato clones with fs abnormality.     

Mutations in AtPS1 (Arabidopsis thaliana Parallel Spindle 1) Lead to the Production of Diploid Pollen Grains

Polyploidy has had a considerable impact on the evolution of many eukaryotes, especially angiosperms. Indeed, most—if not all—angiosperms have experienced at least one round of polyploidy during the course of their evolution, and many important crop plants are current polyploids. The occurrence of 2n gametes (diplogametes) in diploid populations is widely recognised as the major source of polyploid formation. However, limited information is available on the genetic control of diplogamete production. Here, we describe the isolation and characterisation of the first gene, AtPS1 (Arabidopsis thaliana Parallel Spindle 1), implicated in the formation of a high frequency of diplogametes in plants. Atps1 mutants produce diploid male spores, diploid pollen grains, and spontaneous triploid plants in the next generation. Female meiosis is not affected in the mutant. We demonstrated that abnormal spindle orientation at male meiosis II leads to diplogamete formation. Most of the parent''s heterozygosity is therefore conserved in the Atps1 diploid gametes, which is a key issue for plant breeding. The AtPS1 protein is conserved throughout the plant kingdom and carries domains suggestive of a regulatory function. The isolation of a gene involved in diplogamete production opens the way for new strategies in plant breeding programmes and progress in evolutionary studies.     

Triploid planarian reproduces truly bisexually with euploid gametes produced through a different meiotic system between sex

Although polyploids are common among plants and some animals, polyploidization often causes reproductive failure. Triploids, in particular, are characterized by the problems of chromosomal pairing and segregation during meiosis, which may cause aneuploid gametes and results in sterility. Thus, they are generally considered to reproduce only asexually. In the case of the Platyhelminthes Dugesia ryukyuensis, populations with triploid karyotypes are normally found in nature as both fissiparous and oviparous triploids. Fissiparous triploids can also be experimentally sexualized if they are fed sexual planarians, developing both gonads and other reproductive organs. Fully sexualized worms begin reproducing by copulation rather than fission. In this study, we examined the genotypes of the offspring obtained by breeding sexualized triploids and found that the offspring inherited genes from both parents, i.e., they reproduced truly bisexually. Furthermore, meiotic chromosome behavior in triploid sexualized planarians differed significantly between male and female germ lines, in that female germ line cells remained triploid until prophase I, whereas male germ line cells appeared to become diploid before entry into meiosis. Oocytes at the late diplotene stage contained not only paired bivalents but also unpaired univalents that were suggested to produce diploid eggs if they remained in subsequent processes. Triploid planarians may therefore form euploid gametes by different meiotic systems in female and male germ lines and thus are be able to reproduce sexually in contrast to many other triploid organisms.     

MULTIPOLAR SPINDLE 1 (MPS1), a novel coiled-coil protein of Arabidopsis thaliana, is required for meiotic spindle organization

The spindle is essential for chromosome segregation during meiosis, but the molecular mechanism of meiotic spindle organization in higher plants is still not well understood. Here, we report on the identification and characterization of a plant-specific protein, MULTIPOLAR SPINDLE 1 (MPS1), which is involved in spindle organization in meiocytes of Arabidopsis thaliana . The homozygous mps1 mutant exhibits male and female sterility. Light microscopy showed that mps1 mutants produced multiple uneven spores during anther development, most of which aborted in later stages. Cytological analysis showed that chromosome segregation was abnormal in mps1 meiocytes. Immunolocalization showed unequal bipolar or multipolar spindles in mps1 meiocytes, which indicated that aberrant spindles resulted in disordered chromosome segregation. MPS1 encodes a 377-amino-acid protein with putative coiled-coil motifs. In situ hybridization analysis showed that MPS1 is strongly expressed in meiocytes.     

Defects in meiotic chromosome segregation lead to unreduced male gametes in Arabidopsis SMC5/6 complex mutants

Structural maintenance of chromosome 5/6 (SMC5/6) complex is a crucial factor for preserving genome stability. Here, we show that mutants for several Arabidopsis (Arabidopsis thaliana) SMC5/6 complex subunits produce triploid offspring. This phenotype is caused by a meiotic defect leading to the production of unreduced male gametes. The SMC5/6 complex mutants show an absence of chromosome segregation during the first and/or the second meiotic division, as well as a partially disorganized microtubule network. Importantly, although the SMC5/6 complex is partly required for the repair of SPO11-induced DNA double-strand breaks, the nonreduction described here is SPO11-independent. The measured high rate of ovule abortion suggests that, if produced, such defects are maternally lethal. Upon fertilization with an unreduced pollen, the unbalanced maternal and paternal genome dosage in the endosperm most likely causes seed abortion observed in several SMC5/6 complex mutants. In conclusion, we describe the function of the SMC5/6 complex in the maintenance of gametophytic ploidy in Arabidopsis.

Mutants defective in the SMC5/6 complex often fail to divide chromosomes during meiosis, leading to the production of diploid pollen and subsequently triploid offspring.     

Genetics of gamma-irradiation-induced mutations in Arabidopsis thaliana: large chromosomal deletions can be rescued through the fertilization of diploid eggs.

Despite the demonstrated value of chromosomal deletions and deficiencies as tools in plant and animal genome research, in the genetic model plant species Arabidopsis thaliana, such mutations have not been extensively studied. For example, it is not known whether large deletions in different regions of the genome can be tolerated in diploid plants that are heterozygous for such mutations. Similarly the viability or inviability of monosomics has not been examined in detail. To investigate these questions, we have used gamma-irradiated haploid wild-type pollen to pollinate diploid and tetraploid multimarker lines of Arabidopsis. Examination of M1 progenies revealed that chromosome loss mutations and large deletions were induced in the irradiated pollen. Such mutations were eliminated in diploid M1 plants due to dominant lethality but could be rescued in triploid M1 progeny. The use of irradiated pollen and tetraploid marker lines of Arabidopsis is a convenient way of generating deletions and modified chromosomes and provides a genetic tool for deletion mapping and for analysis of chromosomal regions essential for chromosome maintenance.     

How to characterize meiotic functions in plants?

Our understanding of plant meiosis is rapidly increasing thanks to the model Arabidopsis thaliana. Here we present the results of a screening for meiotic mutants carried out with a library containing 30,719 T-DNA insertion lines. An average of one mutant per 1000 lines was recovered. Several phenotypic classes could be distinguished and are presented. In parallel, 39 proteins known to be involved in meiosis in non-plant organisms were chosen and a search was performed for homologue sequences in the completed Arabidopsis thaliana genome. Approximately 30% of the meiotic related sequences showed similarities with one or several Arabidopsis putative genes. The relevance of forward versus reverse genetics in order to characterize meiotic functions is discussed.     

Synaptotagmin1 is required for spindle stability and metaphase-to-anaphase transition in mouse oocytes

Synaptotagmin1, a calcium sensor for exocytosis, forms the 7S complex, or so-called SNARE protein complex, together with SNAP -25, syntaxin and synaptobrevin to mediate docking and fusion of synaptic vesicles to the plasma membrane of the nerve terminal. Here, we identified the unique localization, expression and function of Syt1 during mouse oocyte meiotic maturation by using confocal microscopy, western blotting, Morpholino-based knockdown and time-lapse live cell imaging. We showed that Syt1 expression was gradually increased during oocyte maturation. Syt1 was localized at the oocyte cortex from GV to MII stages and at the spindle poles in MI and MII phases, with one third of a signal-free zone at the oocyte cortex, where the chromosomes are located, which is similar to the distribution pattern of CGs from the pro-MI to MII stages. Knockdown of Syt1 resulted in pro-MI/MI arrest and PB1 extrusion decrease, with severely disrupted spindles and misaligned chromosomes. Knockdown of Syt1 also caused abnormal localization of γ-tubulin, which became redistributed into the cytoplasm. Chromosome spreading showed failure of homologous chromosome segregation. The spindle assembly checkpoint protein Bub3 was detected at the kinetochores even after 10 h of oocyte culture. Live cell imaging analysis revealed that knockdown of Syt1 resulted in abnormal spindles with various morphologies and chromosomes arrested at the pro-MI/MI stage. Defective spindles failed to support chromosome alignment along microtubules, which led to repetitive unsuccessful metaphase-anaphase transitions and failure of PB1 extrusion after extended culture. Taken together, we suggest that Syt1 may act as a MTOC-associated protein to play important roles in mouse oocyte spindle organization/stability, and that it is indispensable for the metaphase-anaphase transition to promote mouse oocyte meiotic maturation.     

Automictic reproduction in interspecific hybrids of poeciliid fish

Automixis, the process whereby the fusion of meiotic products restores the diploid state of the egg, is a common mode of reproduction in plants but has also been described in invertebrate animals. In vertebrates, however, automixis has so far only been discussed as one of several explanations for isolated cases of facultative parthenogenesis. Analyzing oocyte formation in F1 hybrids derived from Poecilia mexicana limantouri and P. latipinna crosses (the cross that led to the formation of the gynogenetic Poecilia formosa), we found molecular evidence for automictic oocyte production. The mechanism involves the random fusion of meiotic products after the second meiotic division. The fertilization of diploid oocytes gives rise to fully viable triploid offspring. Although the automictic production of diploid oocytes as seen in these F1 hybrids clearly represents a preadaptation to parthenogenetic reproduction, it is also a powerful intrinsic postzygotic isolation mechanism because the resulting next generation triploids were always sterile. The mechanism described here can explain facultative parthenogenesis, as well as varying ploidy levels reported in different animal groups. Most importantly, at least some of the reported cases of triploidy in humans can now be traced back to automixis.     

Pollen production and spontaneous polyploidization in diploid populations of Anthoxanthum alpinum

The most common form of polyploidization is that which occurs via the fertilization of unreduced 2 n gametes, i.e. gametes that possess the somatic chromosome number of the species. However, very few data are available concerning the frequency of spontaneous polyploidization in diploid plant populations. In this study we have quantified both the frequency of In pollen production and the frequency of polyploid seed production in diploid populations of the grass Anthoxanthum alpinum. More than 6000 seeds from four different populations collected during two years of study were screened for ploidy using a flow cytometer. In parallel, the frequency both of plants with large pollen (LP) and of LP production on these plants was quantified in two populations. No tetraploid seeds were detected, although in each population a few triploid seeds were regularly produced at a frequency of two triploid seeds per thousand. The frequency of LP producers was similar in the four populations, as was the frequency of LP production. The frequency of LP was ten times that of triploid seeds. These finding suggest that the 'triploid block' is not enough to prevent the regular occurrence of triploids in diploid populations of this species. The role played by the hypothesized 'triploid bridge' in the genesis of autotetraploids is discussed in the light of these results.     

Checkpoint for DNA integrity at the first mitosis after oocyte activation

Activation of oocytes, arrested at the meiosis II (MII) in mammals, initiates meiotic release, mitotic divisions, and development. Unlike most somatic cell types, MII arrested female germ cells lack an efficient DNA integrity checkpoint control. Here we present evidence showing a unique checkpoint for DNA integrity at first mitosis after oocyte activation. Mouse oocytes carrying intact DNA cleaved normally after meiotic release, whereas 50% of oocytes harboring damaged DNA manifested cytofragmentation, a morphological hallmark of apoptosis. If not activated, DNA-damaged MII oocytes did not show apoptotic fragmentation. Further, activated, enucleated oocytes or enucleated fertilized oocytes also underwent cytofragmentation, implicating cytoplasmic coordination of the fragmentation process, independent of the nucleus. Depolymerization of either actin filaments or microtubules induced no cytofragmentation, but inhibited fragmentation upon oocyte activation. During the process of fragmentation, microtubule networks formed, then microtubule asters congregated at discrete locations, around which fragmented cellular bodies formed. Mitotic spindles, however, were not formed inactivated oocytes with damaged or absent DNA; in contrast, normal mitotic spindles were formed in activated oocytes with intact DNA. These results demonstrate that damaged DNA or absence of DNA leads to cytofragmentation after oocyte activation. Further, we found a mechanism of cytoskeletal involvement in the process of cytofragmentation. In addition, possible implication of the present findings in somatic cell cloning and human clinical embryology is discussed.     

Septin 7 is required for orderly meiosis in mouse oocytes

Septin 7 is a conserved GTP-binding protein. In this study, we examined the localization and functions of Septin 7 during mouse oocyte meiotic maturation. Immunofluorescent analysis showed that intrinsic Septin 7 localized to the spindles from the pro-MI stage to the MII stage. Knockdown of Septin 7 by siRNA microinjection caused abnormal spindles and affected extrusion of the first polar body. Septin 7 mRNA tagged with myc was injected into GV stage oocytes to overexpress Septin 7. Overexpressed Myc-Septin 7 localized to the spindle and beneath the plasma membrane displaying long filaments. Fluorescence intensity of spindle α-tubulin in myc-Septin 7-injected oocytes was weaker than that of the control group, demonstrating that Septin 7 may influence recruitment of α-tubulin to spindles. MII oocytes injected with myc-Septin 7 exhibited abnormal chromosome alignment, and parthenogenetic activation failed to allow extrusion of the second polar body, suggesting that overexpression of Septin 7 may affect extrusion of the polar body by disturbing the alignment of chromosomes and regulating α-tubulin recruitment to spindles. In summary, Septin 7 may regulate meiotic cell cycle progression by affecting microtubule cytoskeletal dynamics in mouse oocytes.     

HISTONE DEACETYLASE19 is involved in jasmonic acid and ethylene signaling of pathogen response in Arabidopsis

Histone acetylation is modulated through the action of histone acetyltransferases and deacetylases, which play key roles in the regulation of eukaryotic gene expression. Previously, we have identified a yeast histone deacetylase REDUCED POTASSIUM DEPENDENCY3 (RPD3) homolog, HISTONE DEACETYLASE19 (HDA19) (AtRPD3A), in Arabidopsis thaliana. Here, we report further study of the expression and function of HDA19. Analysis of Arabidopsis plants containing the HDA19:beta-glucuronidase fusion gene revealed that HDA19 was expressed throughout the life of the plant and in most plant organs examined. In addition, the expression of HDA19 was induced by wounding, the pathogen Alternaria brassicicola, and the plant hormones jasmonic acid and ethylene. Using green fluorescent protein fusion, we demonstrated that HDA19 accumulated in the nuclei of Arabidopsis cells. Overexpression of HDA19 in 35S:HDA19 plants decreased histone acetylation levels, whereas downregulation of HDA19 in HDA19-RNA interference (RNAi) plants increased histone acetylation levels. In comparison with wild-type plants, 35S:HDA19 transgenic plants had increased expression of ETHYLENE RESPONSE FACTOR1 and were more resistant to the pathogen A. brassicicola. The expression of jasmonic acid and ethylene regulated PATHOGENESIS-RELATED genes, Basic Chitinase and beta-1,3-Glucanase, was upregulated in 35S:HDA19 plants but downregulated in HDA19-RNAi plants. Our studies provide evidence that HDA19 may regulate gene expression involved in jasmonic acid and ethylene signaling of pathogen response in Arabidopsis.     

Three-dimensional ultrastructure of Saccharomyces cerevisiae meiotic spindles

Meiotic chromosome segregation leads to the production of haploid germ cells. During meiosis I (MI), the paired homologous chromosomes are separated. Meiosis II (MII) segregation leads to the separation of paired sister chromatids. In the budding yeast Saccharomyces cerevisiae, both of these divisions take place in a single nucleus, giving rise to the four-spored ascus. We have modeled the microtubules in 20 MI and 15 MII spindles by using reconstruction from electron micrographs of serially sectioned meiotic cells. Meiotic spindles contain more microtubules than their mitotic counterparts, with the highest number in MI spindles. It is possible to differentiate between MI versus MII spindles based on microtubule numbers and organization. Similar to mitotic spindles, kinetochores in either MI or MII are attached by a single microtubule. The models indicate that the kinetochores of paired homologous chromosomes in MI or sister chromatids in MII are separated at metaphase, similar to mitotic cells. Examination of both MI and MII spindles reveals that anaphase A likely occurs in addition to anaphase B and that these movements are concurrent. This analysis offers a structural basis for considering meiotic segregation in yeast and for the analysis of mutants defective in this process.     

The Microtubule-Associated Protein ASPM Regulates Spindle Assembly and Meiotic Progression in Mouse Oocytes

The microtubule-associated protein ASPM (abnormal spindle-like microcephaly-associated) plays an important role in spindle organization and cell division in mitosis and meiosis in lower animals, but its function in mouse oocyte meiosis has not been investigated. In this study, we characterized the localization and expression dynamics of ASPM during mouse oocyte meiotic maturation and analyzed the effects of the downregulation of ASPM expression on meiotic spindle assembly and meiotic progression. Immunofluorescence analysis showed that ASPM localized to the entire spindle at metaphase I (MI) and metaphase II (MII), colocalizing with the spindle microtubule protein acetylated tubulin (Ac-tubulin). In taxol-treated oocytes, ASPM colocalized with Ac-tubulin on the excessively polymerized microtubule fibers of enlarged spindles and the numerous asters in the cytoplasm. Nocodazole treatment induced the gradual disassembly of microtubule fibers, during which ASPM remained colocalized with the dynamic Ac-tubulin. The downregulation of ASPM expression by a gene-specific morpholino resulted in an abnormal meiotic spindle and inhibited meiotic progression; most of the treated oocytes were blocked in the MI stage with elongated meiotic spindles. Furthermore, coimmunoprecipitation combined with mass spectrometry and western blot analysis revealed that ASPM interacted with calmodulin in MI oocytes and that these proteins colocalized at the spindle. Our results provide strong evidence that ASPM plays a critical role in meiotic spindle assembly and meiotic progression in mouse oocytes.     

AtSPO11-1 is necessary for efficient meiotic recombination in plants

The Saccharomyces cerevisiae Spo11 protein catalyses DNA double-strand breaks (DSBs) that initiate meiotic recombination. The model plant Arabidopsis thaliana possesses at least three SPO11 homologues. T-DNA and ethyl-methane sulfonate mutagenesis allowed us to show that meiotic progression is altered in plants in which the AtSPO11-1 gene is disrupted. Both male and female meiocytes formed very few bivalents. Furthermore, no fully synapsed chromosomes were observed during prophase I. Later, in meiosis I, we observed that chromosomes segregated randomly, leading to the production of a large proportion of non-functional gametes. These meiotic aberrations were associated with a drastic reduction in meiotic recombination. Thus, our data show that initiation of meiotic recombination by SPO11- induced DSBs is a mechanism conserved in plants. Furthermore, unlike Drosophila and Caenorhabditis elegans, but like fungi, SPO11 is necessary for normal synapsis in plants.     

Variation in pollen formation and its cytological mechanism in an allotriploid white poplar

Variation in pollen formation and its cytological mechanism in an allotriploid white poplar were investigated by the squashed technique and indirect immunofluorescence. Besides 0.5% stuck pollen grains, this allotriploid produced regularly spherical pollen grains. It was estimated that 90.3% of pollen grains were viable. Diameters of the viable spherical pollen grains ranged from 23.2 to 72.9 μm, with a bimodal frequency distribution. Numerous meiotic abnormalities were found, including highly irregular chromosome pairing, lagging chromosomes and chromosome bridges, micronuclei, and multiple spindles, which indicate highly genetic imbalance of this allotriploid. Some micronuclei triggered minispindle formation in metaphase II and participated in cytokinesis to form microcytes in sporads. Abnormal orientation of metaphase II spindles contributed to production of dyads and triads, which produced unreduced microspores. However, parallel orientation of spindles was not necessary for dyad formation, because an organelle band positioned in the equatorial region prevented the spindles from coalescing. Some microsporocytes exhibited a complete or partial absence of cytokinesis, which resulted in the formation of monads and the increased frequency of dyads and triads. The perspective of this triploid in the polyploid breeding program of white poplar is discussed.     

Polyploidy‐associated genomic instability in Arabidopsis thaliana

Formation of polyploid organisms by fertilization of unreduced gametes in meiotic mutants is believed to be a common phenomenon in species evolution. However, not well understood is how species in nature generally exist as haploid and diploid organisms in a long evolutionary time while polyploidization must have repeatedly occurred via meiotic mutations. Here, we show that the ploidy increased for two consecutive generations due to unreduced but viable gametes in the Arabidopsis cyclin a1;2‐2 (also named tardy asynchronous meiosis‐2) mutant, but the resultant octaploid plants produced progeny of either the same or reduced ploidy via genomic reductions during meiosis and pollen mitosis. Ploidy reductions through sexual reproduction were also observed in independently generated artificial octaploid and hexaploid Arabidopsis plants. These results demonstrate that octaploid is likely the maximal ploidy produced through sexual reproduction in Arabidopsis. The polyploidy‐associated genomic instability may be a general phenomenon that constrains ploidy levels in species evolution. genesis 48:254–263, 2010. © 2010 Wiley‐Liss, Inc.     

Changes in genomic methylation patterns during the formation of triploid asexual dandelion lineages

DNA methylation is an epigenetic mechanism that has the potential to affect plant phenotypes and that is responsive to environmental and genomic stresses such as hybridization and polyploidization. We explored de novo methylation variation that arises during the formation of triploid asexual dandelions from diploid sexual mother plants using methylation‐sensitive amplified fragment length polymorphism (MS‐AFLP) analysis. In dandelions, triploid apomictic asexuals are produced from diploid sexual mothers that are fertilized by polyploid pollen donors. We asked whether the ploidy level change that accompanies the formation of new asexual lineages triggers methylation changes that contribute to heritable epigenetic variation within novel asexual lineages. Comparison of MS‐AFLP and AFLP fragment inheritance in a diploid × triploid cross revealed de novo methylation variation between triploid F₁ individuals. Genetically identical offspring of asexual F₁ plants showed modest levels of methylation variation, comparable to background levels as observed among sibs in a long‐established asexual lineage. Thus, the cross between ploidy levels triggered de novo methylation variation between asexual lineages, whereas it did not seem to contribute directly to variation within new asexual lineages. The observed background level of methylation variation suggests that considerable autonomous methylation variation could build up within asexual lineages under natural conditions.