Active role of lagging chromosomes in spindle collapse as revealed by live phase contrast and tubulin immunostaining in grasshopper spermatocytes

Univalents, that is, chromosomes lacking an attached partner at the first meiotic division, show extremely faulty transmission. Most segregational errors stem from amphitelic (mitotic-like) orientation at metaphase I followed by anaphase I lagging. Our studies in living grasshopper spermatocytes show that amphitelic orientation may provoke spindle collapse: spindle elongation and cytokinesis are impaired and an unreduced restitution nucleus is formed. This does not prevent meiotic progression and eventually leads to the production of diploid gametes. The morphology and characteristics of spindle collapse in our material, as revealed by in vivo observation and tubulin immunostaining, indicate an active role of the chromosomes in the whole process.     

Abnormal macrosporogenesis in five alfalfa (Medicago sativa) mutants producing 4n pollen

Summary An analysis of micro- and macrosporogenesis in five diploid alfalfa mutants was carried out using a stain-clearing technique. All plants produced tetranucleated microspores and jumbo pollen due to the complete failure of the postmeiotic cytokinesis as well as bi- and trinucleated macrospores. The latter was due to the absence of cytokinesis after the first and second meiotic division of macrosporogenesis. Only one out of the five clones analyzed formed tetranucleated macrospores as a consequence of the total lack of cytokinesis after both meiotic divisions. The fusion of nuclei within binucleated macrospores resulted in 2n macrospores of the SDR type, recognizable on the basis of nucleolus dimension, confirming the ability of jumbo pollen (jp) mutants to produce 2n eggs at a high frequency. Nuclear fusion was also observed within tri- and tetranucleated macrospores. Although having the same genetic background, the five clones showed significant variability in the expression of abnormal cytokinesis during macrosporogenesis.Research supported by National Research Council of Italy, special project RAISA, sub-project no. 2, paper no. 527     

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.     

Aberrant microspore development in hybrids of maize x Tripsacum dactyloides.

Cytogenetic investigations of meiosis in hybrids between maize and Tripsacum have been well documented; however, the inherent problem of male and female sterility has not been addressed either on a genetic or cytogenetic level. The purpose of this cytological study was to identify some of the probable causes of male sterility in maize x Tripsacum dactyloides hybrids. Disturbances in pollen development of maize x T. dactyloides hybrids, derived from both diploid (2n) and tetraploid (4n) Tripsacum sources, were commonly observed. Anomalies in the development of the microspore apparently occurred because of a failure of the chromosomes to congregate at the metaphase plate, development of a tripolar spindle, and failure of cytokinesis at the first and second meiotic divisions. Phenotypic features of abnormal microspore development were the maturation of large pollen grains, "Siamese" pollen grains, the occurrence of variable invaginations, and a nuclear budding-type behavior. These abnormalities were not observed in the 56-chromosome amphidiploid or the 38-chromosome backcross generations.     

AFLP-based estimation of 2n gametophytic heterozygosity in two parthenogenetically derived dihaploids of Rosa hybrida L

Two dihaploid Rosa hybrida L. genotypes, derived through parthenogenesis by using irradiated pollen, were crossed with clonally propagated plants of the diploid species Rosa rugosa Thunb. and Rosa wichuraiana Crép., respectively. Three progeny groups were obtained which contained numerous polyploids, as determined by flow cytometry. Production of fertile 2n female gametes is apparently very common in one of these R. hybrida dihaploid derivatives, whereas the other one is able to produce fertile 2n pollen. Hence, an amplified fragment length polymorphism (AFLP) study was performed on the parental plants and the resulting hybrid offspring in order to estimate (1) the respective genomic parental contributions, and (2) the level of heterozygosity transmitted by the 2n unreduced gametes. Comparison of the levels of transmitted parental heterozygosity revealed that two types of 2n gametes were produced simultaneously, presumably resulting from restitution at the first and at the second meiotic division, respectively. Received: 15 February 2001 / Accepted: 22 May 2001     

Genetics of the Tubulin Gene Families of Physarum

The organization of the alpha- and beta-tubulin gene families in Physarum was investigated by Mendelian analysis. Restriction endonuclease-generated DNA fragments homologous to alpha- and beta-tubulin show length polymorphisms that can be used as markers for genetic mapping. Analysis of meiotic assortment among progeny of heterozygotes allowed alpha- and beta-tubulin sequence loci to be defined. There are four unlinked alpha-tubulin sequence loci (altA, altB, altC and altD) and at least three unlinked beta-tubulin sequence loci (betA, betB and betC). The alpha-tubulin loci are not linked to the beta-tubulin loci. --Segregation of tubulin sequence loci with respect to ben mutations that confer resistance to antitubulin benzimidazole drugs was used to investigate whether any members of the alpha- or beta-tubulin gene families are allelic to ben loci. The beta-tubulin sequence locus betB is allelic to the resistance locus benD, the betA locus is probably allelic to benA and the alpha-tubulin sequence locus altC may be allelic to benC. The molecular implications of benzimidazole resistance phenotypes when only one of the expressed beta-tubulin gene family members mutates to drug resistance are discussed in relation to tubulin function.     

A dual effect on protein synthesis and degradation modulates the tubulin level in rice cells treated with oryzalin

The effect of the anti-microtubular drug oryzalin on growth and morphology of cultured rice (Oryza sativa L., cv. Roncarolo) cells was evaluated with specific reference to mechanisms that control intracellular tubulin levels. The addition of oryzalin caused a great reduction in the level of both alpha- and beta-tubulin polypeptides, as detected by Western blot analysis. However, no appreciable decrease was observed in the population of total or isotype-specific alpha- and beta-tubulin mRNAs. Only within the first 24 h of the oryzalin treatment, when the level of both alpha- and beta-tubulin polypeptides was still undiminished, was a consistent reduction in the amount of total beta-tubulin mRNA observed. Pulse-chase experiments performed on rice cells grown in the presence of 1 microM oryzalin revealed the presence of two distinct mechanisms that negatively control alpha- and beta-tubulin polypeptide levels. (i) There was an immediate effect on protein synthesis, which resulted in a reduction in the level of newly synthesized tubulin. (ii) There was a delayed response characterized by a substantial degradation of both alpha- and beta-tubulin monomers; this degradation occurred after 24 h of herbicide treatment. The possible involvement of Ca2+ in the degradation of the unincorporated tubulin monomers is also documented and discussed.     

Chromosome numbers and meiotic analysis in the pre-breeding of <Emphasis Type="BoldItalic">Brachiaria decumbens</Emphasis> (Poaceae)

A total of 44 accessions of Brachiaria decumbens were analysed for chromosome count and meiotic behaviour in order to identify potential progenitors for crosses. Among them, 15 accessions presented 2n = 18; 27 accessions, 2n = 36; and 2 accessions, 2n = 45 chromosomes. Among the diploid accessions, the rate of meiotic abnormalities was low, ranging from 0.82% to 7.93%. In the 27 tetraploid accessions, the rate of meiotic abnormalities ranged from 18.41% to 65.83%. The most common meiotic abnormalities were related to irregular chromosome segregation, but chromosome stickiness and abnormal cytokinesis were observed in low frequency. All abnormalities can compromise pollen viability by generating unbalanced gametes. Based on the chromosome number and meiotic stability, the present study indicates the apomictic tetraploid accessions that can act as male genitor to produce interspecific hybrids with B. ruziziensis or intraspecific hybrids with recently artificially tetraploidized accessions.     

Crossing over and Diploid Egg Formation in the Elongate Mutant of Maize

Rhoades (1941) found recombination in the proximal regions of chromosome 5 to be higher in male than in female flowers. Two explanations were proposed to account for the lower female values, namely: (1) there is a basic difference in rates of crossing over in mega- and microsporocytes, or (2) selective orientation of the chromosome 5 bivalent on the meiotic spindle leads to the preferential segregation of noncrossover chromatids to the basal megaspore. These alternatives have been tested by carrying out a half-tetrad analysis of the diploid eggs produced by plants homozygous for the recessive elongate (el) allele. The A2-Bt crossover values determined from the diploid eggs of elongate plants were much lower than those calculated from haploid sperm of both El el and el el plants. Since male and female flowers should have similar cross-over values if the orientation hypothesis were correct, it was concluded that the amount of crossing over in the A2-Bt region of chromosome 5 is intrinsically higher in male than in female meiocytes. In the analysis of diploid eggs the use of the Bt locus, which marks the centric region of chromosome 5, provided information on the origin of diploid eggs. The genotypic constitution of 425 diploid eggs was ascertained. Of these, 20.4% were Bt bt. They could not be accounted for by failure of the second meiotic division or by replication during the interphase between the two meiotic divisions, but are expected if there is a single division with an equational separation of the centromere regions of chromosome 5. The Bt Bt and bt bt genotypes arise from a disjunctional separation. It is proposed that diploid eggs are produced by an abnormal meiosis in which there is one division with either disjunctional or equational separation. Disjunctional separation is more frequent but the ratio of the two types varies from ear to ear. Recombination in the A2-Bt-Pr region of chromosome 5 was found to be higher in the haploid gametes of elongate homozygotes than in El El and El el plants. On the other hand, crossing over was reduced in the Sh-Bz segment of chromosome 9 in elongate plants, but the adjacent Bz-Wx interval was unaffected.     

Genetic dissection of meiotic cytokinesis in Drosophila males

We have used Drosophila male meiosis as a model system for genetic dissection of the cytokinesis mechanism. Drosophila mutants defective in meiotic cytokinesis can be easily identified by their multinucleate spermatids. Moreover, the large size of meiotic spindles allows characterization of mutant phenotypes with exquisite cytological resolution. We have screened a collection of 1955 homozygous mutant male sterile lines for those with multinucleate spermatids, and thereby identified mutations in 19 genes required for cytokinesis. These include 16 novel loci and three genes, diaphanous, four wheel drive, and pebble, already known to be involved in Drosophila cytokinesis. To define the primary defects leading to failure of cytokinesis, we analyzed meiotic divisions in male mutants for each of these 19 genes. Examination of preparations stained for tubulin, anillin, KLP3A, and F-actin revealed discrete defects in the components of the cytokinetic apparatus, suggesting that these genes act at four major points in a stepwise pathway for cytokinesis. Our results also indicated that the central spindle and the contractile ring are interdependent structures that interact throughout cytokinesis. Moreover, our genetic and cytological analyses provide further evidence for a cell type-specific control of Drosophila cytokinesis, suggesting that several genes required for meiotic cytokinesis in males are not required for mitotic cytokinesis.     

Specific inhibition of endogenous beta-tubulin synthesis in Xenopus oocytes by anti-messenger oligodeoxynucleotides

An oligodeoxynucleotide containing 27 nucleotides, complementary to a highly conserved sequence of beta-tubulin mRNAs, led to a nearly complete inhibition of beta-tubulin synthesis in Xenopus oocytes after microinjection. Inhibition persisted 24 hours post-injection and was specific for beta-tubulin as the synthesis of alpha-tubulin as well as that of other proteins from the oocyte was not affected. Complete inhibition of beta-tubulin synthesis did not prevent progesterone-induced meiotic maturation and formation of the chromosome spindle. This result indicates that the pool of endogenous tubulin already present in fully-grown oocytes is sufficient to allow normal meiotic maturation. This finding correlates with previous experiments showing that the turn-over of tubulin is very slow in the oocyte.     

Meiotic behavior as a selection tool in silage corn breeding

In breeding programs, commercial hybrids are frequently used as a source of inbred lines to obtain new hybrids. Considering that maize production is dependent on viable gametes, the selection of populations to obtain inbred lines with high meiotic stability could contribute to the formation of new silage corn hybrids adapted to specific region. We evaluated the meiotic stability of five commercial hybrids of silage corn used in southern Brazil with conventional squashing methods. All of them showed meiotic abnormalities. Some abnormalities, such as abnormal chromosome segregation and absence of cytokinesis, occurred in all the genotypes, while others, including cytomixis and abnormal spindle orientation, were found only in some genotypes. The hybrid SG6010 had the lowest mean frequency of abnormal cells (21.27%); the highest frequency was found in the hybrid P30K64 (44.43%). However, the frequency of abnormal meiotic products was much lower in most genotypes, ranging from 7.63% in the hybrid CD304 to 43.86% in Garra. Taking into account the percentage of abnormal meiotic products and, hence, meiotic stability, only the hybrids CD304, P30K64, SG6010, and P30F53 are recommended to be retained in the breeding program to obtain inbred lines to create new hybrids.     

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.     

Microsporogenesis in Brachiaria brizantha (Poaceae) as a selection tool for breeding

The genus Brachiaria comprises more than 100 species and is the single most important genus of forage grass in the tropics. Brachiaria brizantha, widely used in Brazilian pastures for beef and dairy production, is native to tropical Africa. As a subsidy to the breeding program underway in Brazil, cytological studies were employed to determine the chromosome number and to evaluate microsporogenesis in 46 accessions of this species available at Embrapa Beef Cattle (Brazil). Thirty-four accessions presented 2n = 36; seven had 2n = 45, and five had 2n = 54 chromosomes. Based on the higher level of chromosome association observed in diakinesis, in tetra-, penta-, and hexavalents, respectively, it was concluded that they are derived from x = 9; consequently, these accessions are tetra- (2n = 4x = 36), penta- (2n = 5x = 45), and hexaploids (2n = 6x = 54). The most common meiotic abnormalities were irregular chromosome segregation due to polyploidy. Chromosome stickiness, abnormal cytokinesis, non-congressed bivalents in metaphase I and chromosomes in metaphase II, and chromosome elimination were recorded at varying frequencies in several accessions. The mean percentage of meiotic abnormalities ranged from 0.36 to 95.76%. All the abnormalities had the potential to affect pollen viability by generating unbalanced gametes. Among the accessions, only the tetraploid ones with less than 40% of abnormalities are suitable as pollen donors in intra- and interspecific crosses. Currently, accessions with a high level of ploidy (5 and 6n) cannot be used as male genitors in crosses because of the lack of sexual female genitors with the same levels of ploidy.     

Polyglycylation of tubulin is essential and affects cell motility and division in Tetrahymena thermophila

We analyzed the role of tubulin polyglycylation in Tetrahymena thermophila using in vivo mutagenesis and immunochemical analysis with modification-specific antibodies. Three and five polyglycylation sites were identified at glutamic acids near the COOH termini of alpha- and beta-tubulin, respectively. Mutants lacking all polyglycylation sites on alpha-tubulin have normal phenotype, whereas similar sites on beta-tubulin are essential. A viable mutant with three mutated sites in beta-tubulin showed reduced tubulin glycylation, slow growth and motility, and defects in cytokinesis. Cells in which all five polyglycylation sites on beta-tubulin were mutated were viable if they were cotransformed with an alpha-tubulin gene whose COOH terminus was replaced by the wild-type COOH terminus of beta-tubulin. In this double mutant, beta-tubulin lacked detectable polyglycylation, while the alpha-beta tubulin chimera was hyperglycylated compared with alpha-tubulin in wild-type cells. Thus, the essential function of polyglycylation of the COOH terminus of beta-tubulin can be transferred to alpha-tubulin, indicating it is the total amount of polyglycylation on both alpha- and beta-tubulin that is essential for survival.     

Dynamics of microtubular cytoskeleton in higher plant meiosis. V. Late prometaphase. The general scheme of anastral spindle formation

By means of morphological analyses of meiotic abnormalities in pollen mother cells (PMCs) of cereal wide hybrids, haploids and meiotic mutants, the processes involved in cytoskeleton cycle at late prometaphase (a sub-stage of transition from chaotic figure to bipolar spindle) were studied. A significance of the four processes of late prometaphase--axial orientation, lateral association, consolidation and convergance of spindle fibers--is discussed.     

Meiotic irregularities in diploid and tetraploid plants of <Emphasis Type="Italic">Lolium multiflorum</Emphasis> Lam.

The genus Lolium comprises several species of economical importance in temperate countries, mostly due to their high nutritional value and adaptability to cold environments, including southern regions of Brazil. Recently, several diploid cultivars and populations, as well as synthetic tetraploid cultivars have been explored. In order to viabilize or to direct crossings, it is important that the accessions present regular meiosis, thus, producing viable pollen grains. In this way, this study aimed at investigating the meiosis of nine accessions of Lolium multiflorum used in breeding programs, seeking to evaluate its viability in future crossings. The meiosis of diploid plants (2n?=?2×?=?14) is more regular than the artificially tetraploidized genotypes (2n?=?4×?=?28). In the tetraploids, univalent, bivalent, and multivalent configurations were observed. The irregularities were associated to mixoploidy, irregular segregation of chromosomes, spindle disorders, asynchrony, micronuclei, and cellular fusion and formation of syncyte. The abnormalities affected the meiotic index of tetraploid cultivars, characterizing them as unstable. Nevertheless, both diploid and tetraploid genotypes are considered able to participate in crossings because, although there are abnormalities, they do not occur at levels that could affect the production of viable pollen grains.     

Tubulin composition and microtubule nucleation of a griseofulvin-resistant Chinese hamster ovary cell mutant with abnormal spindles

A griseofulvin-resistant Chinese hamster ovary (CHO) mutant (Grs-2) which has an altered beta-tubulin subunit as well as wild-type beta-tubulin is temperature-sensitive (ts) for growth at 40.5 degrees C. This growth defect appears to result from the formation of abnormal mitotic spindles at the non-permissive temperature (Abraham, I et al., J cell biol 97 (1983) 1055) [19]. Light microscopy of spindles isolated from mutant cells cultured at the permissive temperature showed a typical bipolar morphology, whereas spindles isolated at the non-permissive temperature were multipolar. In order to study the role of tubulin in spindle formation, we analyzed the tubulin composition of the multipolar spindles. Two-dimensional gels and immunoblotting analysis of one-dimensional electrophoretic gels stained with monoclonal anti-Chinese hamster brain beta-tubulin antibody revealed that both mutant and wild-type beta-tubulins were present in similar proportions in both bipolar spindles at 37 degrees C and multipolar spindles at 40.5 degrees C. The ratio between wild-type and mutant tubulin in spindles was also found to be the same as in the cytoplasmic microtubule network in interphase cells, providing evidence that the mutant beta-tubulin appeared to be incorporated in a similar manner into both interphase and mitotic microtubule structures. In vitro microtubule polymerization onto centrosomes prepared from mutant Grs-2 demonstrated that 80% of the sites for microtubule nucleation were without centrioles, suggesting fragmentation of pericentriolar material away from centrioles. This may be one of the causes of multipolar spindle formation in the mutant cells. These results, therefore, suggest that abnormal formation of spindles in mutant cells is due not to the presence of the mutant tubulin per se, but to the abnormal behavior of this mutant tubulin in the cellular environment during mitosis or abnormal interaction with other components in the spindle at 40.5 degrees C.     

The tubulin genes of Trypanosoma cruzi

The organization of the alpha- and beta-tubulin genes in the genome of Trypanosoma cruzi have been analysed by Southern blotting using tubulin probes derived from Trypanosoma brucei. The tubulin array appears to be more complex in this organism than in other members of the same family. Some tubulin genes are tightly clustered in an alternating (alpha-beta)n array with a basic repeat unit length of 4.3 kb. However, other pairs of alternating alpha- and beta-tubulin sequences appear to be physically separated from the basic group. This finding indicates that the tubulin gene cluster present in T. cruzi is less perfectly conserved than in T. brucei. T. (Herpetosoma) rangeli is similar to T. (Schizotrypanum) cruzi in its tubulin gene organization whereas most of these genes are tandemly clustered in the genome of T. (Trypanozoon) evansi, with a basic repeat unit length of 3.6 kb as previously described for T. (Trypanozoon) brucei. Two overlapping recombinant clones containing T. cruzi tubulin sequences have been isolated from a genomic cosmid library of T. cruzi epimastigotes using the T. brucei tubulin probes. Partial sequencing of the T. cruzi beta-tubulin gene has confirmed its identity and shows more than 70% homology with the sea urchin, chicken and T. b. rhodesiense beta-tubulin reported gene sequences. Analysis of tubulin gene organization through the parasite life cycle does not show evidence of major rearrangements within the repeat unit. Several T. cruzi strains and cloned lines whilst sharing the 4.3-kb tubulin repeat unit, exhibited very variable tubulin gene organization with tubulin probes. These striking differences in the organization of this structural gene among T. cruzi strains and cloned lines suggest that the heterogeneity previously reported in parasite populations may be related to a very dynamic, diploid genome.