The dsy10 Mutation of Arabidopsis results in desynapsis and a general breakdown in meiosis

The proper pairing and recombination of chromosomes during prophase is essential for the formation of gametes during meiosis. As part of studies to identify genes required for homologous chromosome pairing and recombination during meiosis in plants, we characterized a number of T-DNA-tagged, male-sterile mutants of Arabidopsis. Preliminary cytological studies on one line, 7219 which is male and female sterile, suggested that the mutation may disrupt meiosis and result in the formation of aberrant microsporocytes and microspores. In this report we present the results of a detailed analysis of meiosis in microsporocytes of sterile plants to elucidate the nature of the 7219 mutation. Analysis indicates that the mutation usually results in a desynaptic phenotype, with ten sister chromatids observed prior to metaphase I in most cells. Based on this, we named the mutation dsy10. The presence of several other meiotic defects suggests that dsy10 may not be a typical desynaptic mutant. Received: 15 December 2000 / Accepted: 19 April 2001     

Immunocytochemical visualization of the centromeres during male and female meiosis in Lilium longiflorum

Immunofluorescence staining with an antiserum raised against a presumptive meiotic histone, which has been shown to appear prior to male meiosis in liliaceous plants, preferentially stained the centromere (kinetochore) region of meiotic chromosomes in microsporocytes and megasporocytes. Using this antiserum, we were able clearly to visualize the centromeres at all important meiotic stages in microsporocytes, namely, the association and fusion of centromeres of homologous chromosomes at zygotene-pachytene in prophase I, the disjunction of the homologous centromeres at diplotene, the doubling of each centromere at metaphase I and nonseparation of the sister centromeres at anaphase I, by confocal laser scanning microscopy. Thus, this report provides a complete picture of the behavior of centromeres during meiosis in a eukaryote for the first time. This antiserum also decorated centromeres during female meiosis in cryo-sectioned megasporocytes, but did not stain the centromeres of mitotic chromosomes in root-tip meristem. From these observations, it is suggested that a meiosis-specific centromere protein is required for the meiosis-specific behavior of the centromere. Received: 12 May 1997; in revised form: 20 August 1997 / Accepted: 25 August 1997     

Evidence for separate genetic control of crossing over and chiasma maintenance in maize

The meiotic cytological behavior of chromosomes in maize microsporocytes homozygous for the recessive mutant desynaptic was studied at various stages. It was found that following apparently normal pachytene synapsis there appears to be sporadic precocious desynapsis. By diakinesis bivalents heterozygous for a distal knob have often separated to pairs of univalents, each with a knob-carrying and a knobless chromatid. From the frequency of such events it is inferred that the crossover process is probably not affected by the mutant and that the genetic defect affects instead a distinct function concerned with chiasma maintenance following crossing over. Since precocious separation of dyads to monads at prophase II was also found in the desynaptic material, it is suggested that normal chiasma maintenance until anaphase I and normal dyad integrity maintenance between anaphase I and anaphase II may depend upon the same mechanism; it is also suggested that this may involve a special tendency for cohesiveness of sister chromatids during meiosis, beyond that which is ordinarily found at mitosis.     

The inhibition of protein synthesis in meiotic cells and its effect on chromosome behavior

Autoradiographs show that tritiated leucine is incorporated into protein continually at an almost linear rate during meiotic prophase of lily microsporocytes in in vitro culture. Although label is mostly in the cytoplasm for the first hour, it becomes almost evenly distributed throughout the cell after a few hours. The amount of label decreases slightly, if at all, during a chase period extending through the rest of the prophase — a period of 3 to 4 days. — The incorporation of label was blocked by 95% by the protein inhibitor, cycloheximide, at a concentration of 3.5 × 10^-6 M. In the presence of this inhibitor, meiosis was arrested at all stages through metaphase I and even later. After temporary inhibition, however, or in low drug concentrations, characteristic cytological abnormalities subsequently developed, depending on the meiotic stage at which the inhibition occurred. One important observation was that the formation of chiasmata between homologs could be blocked if the inhibition was applied during the late zygotene or early pachytene stages.This work was supported by a grant from the National Science Foundation (GB-5173 X).USPHS postdoctoral fellow.     

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 comparative study of male meiosis in Drosophila melanogaster and D. virilis

Male meiosis in D. melanogaster cytologically follows the usual pattern, whereas in D. melanogaster and in D. virilis oocytes the chromosomes clump into a karyosphere at early meiotic prophase and remain so up to metaphase I.Male meiosis in D. virilis spermatocytes has an intermediate character: a part of the chromatin clumps together in a karyosphere at early prophase, whereas the other part of the chromatin remains diffuse all through prophase. At the end of prophase, the diffuse chromatin becomes integrated into the karyosphere before metaphase I. During the meiotic divisions the chromosomes have the same clumped aspect as those in Drosophila oocytes and thus differ strikingly from the dividing chromosomes in D. melanogaster spermatocytes.In D. virilis spermatocytes the nucleolus exhibits changes during the meiotic prophase that may be related to synthetical activities. The DNA specific staining with the fluorochrome DAPI reveals the existence of extrachromosomal DNA in the later prophase. Other striking differences in meiotic events between the two Drosophila species concern the centrioles and spermiogenesis.     

AUTORADIOGRAPHIC STUDIES OF NUCLEIC ACIDS AND PROTEINS DURING MEIOSIS IN LILIUM LONGIFLORUM

Taylor , J. Herbert (Columbia U., New York, N. Y.) Autoradiographic studies of nucleic acids and proteins during meiosis in Lilium longiflorum. Amer. Jour. Bot. 46(7): 477–484. Illus. 1959.—A study was made of the incorporation of glycine-C¹⁴, orotic acid-C¹⁴ and cytidine-H³ into nucleic acids and proteins of sporogenous and tapetal cells of lily anthers preceding and during meiosis. Methods for differential extraction of nucleic acids from tissue sections, which had been frozen, dehydrated by alcohol-substitution, and fixed in hot alcohol, were tested by chromatographic analysis of extracts. Both acid and enzyme hydrolysis were shown to be useful for quantitative or, at least, semi-quantitative work. DNA synthesis was shown to occur only during premeiotic interphase in sporogenous cells, but at two intervals in tapetal nuclei, once when the microsporocytes are in zygotene and again during pachytene. Each time the synthetic period was followed by a normal mitosis. Accumulation of RNA in microsporocytes occurred at stages up to late leptotene. After this period, labeled RNA accumulated almost exclusively in their nuclei and at a slower rate than in earlier stages. DNA synthesis, as measured by incorporation of glycine-C¹⁴ and orotic acid-C¹⁴, gave the same results and confirm earlier results with inorganic phosphate-P³². For RNA, glycine-C¹⁴ and orotic acid-C¹⁴ gave different results. When glycine-C¹⁴ was the source of label, incorporation of C¹⁴ in RNA stopped during DNA synthesis in sporogenous cells. Glycine-C¹⁴ was not utilized to a significant extent at any time by tapetal cells for RNA synthesis, but extensively for DNA and protein synthesis. Orotic acid-C¹⁴ was incorporated into RNA of both tapetum and sporogenous cells at various periods in development apparently including the interval of DNA synthesis. Protein synthesis as measured by incorporation of glycine is relatively rapid during premeiotic interphase and leptotene. It continues during the remainder of prophase, but at a much reduced rate. In tapetal cells the rate is rapid in the nuclei during periods of DNA synthesis, but even faster in both cytoplasm and nucleus after divisions are completed and the microsporocytes are in late prophase and division stages. This period of synthesis is perhaps necessary for the postmeiotic functioning of tapetum when it appears to secrete the wall materials for the microspores.     

The<Emphasis Type="Italic">atspo11-1</Emphasis> mutation rescues <Emphasis Type="Italic">atxrcc3</Emphasis> meiotic chromosome fragmentation

Homologous recombination events occurring during meiotic prophase I ensure the proper segregation of homologous chromosomes at the first meiotic division. These events are initiated by programmed double-strand breaks produced by the Spo11 protein and repair of such breaks by homologous recombination requires a strand exchange activity provided by the Rad51 protein. We have recently reported that the absence of AtXrcc3, an ArabidopsisRad51 paralogue, leads to extensive chromosome fragmentation during meiosis, first visible in diplotene of meiotic prophase I. The present study clearly shows that this fragmentation results from un- or mis-repaired AtSpo11-1 induced double-strand breaks and is thus due to a specific defect in the meiotic recombination process.     

Histone acetylation during meiosis in Lilium microsporocytes

Acetate incorporation into histones of Lilium microsporocytes is consistently higher in early prophase of meiosis than in later stages. The pattern of acetate incorporation into histones differs from that found for the soluble nuclear proteins. Evidence is presented that acetate incorporation reflects true acetylation, not histone synthesis. e-N-acetyllysine (eNAcLys) was released from histones by enzymatic digestion. Amino acid analysis of the digests confirm the presence of significantly higher amounts of eNAcLys in early meiotic stages. These results are consistent with the hypothesis that modulation of histone charge plays a role in the binding of histones to DNA and/or chromosome condensation. In vitro levels of histone acetylating activity remain constant throughout meiosis. The results suggest that microsporocyte histone deacetylase activity increases through meiotic prophase.     

Cytological analysis of MRE11 protein during early meiotic prophase I in Arabidopsis and tomato

Early recombination nodules (ENs) are multiprotein complexes that are thought to be involved in synapsis and recombination, but little is known about their components or how they may be involved in these events. In this study, we describe the cytological behavior of a possible EN component, MRE11, a protein that is important for the repair of the numerous, programmed deoxyribonucleic acid double-strand breaks (DSBs) that occur early in the meiotic prophase. By immunofluorescence, many MRE11 foci were associated with chromosomal axes during early prophase I in both wild-type Arabidopsis and tomato primary microsporocytes. Similar patterns of MRE11 foci were observed in two Arabidopsis mutants (Atspo11-1 and Atprd1) that are defective in DSB formation and synapsis. In tomato chromosomes, MRE11 foci were more common in distal euchromatin than in proximal heterochromatin, consistent with known EN patterns. However, electron microscopic immunogold localization demonstrated that only about 10% of ENs were labeled, and most MRE11 label was associated with synaptonemal complex components. Thus, in plants, MRE11 foci are not dependent on DSB formation, and most MRE11 foci do not correspond to ENs. More generally, our results show that the simple presence of large numbers of fluorescent foci associated with synapsing chromosomes is insufficient evidence to equate these foci with ENs. Electronic supplementary material The online version of this article (doi:) contains supplementary material, which is available to authorized users. Leslie D. Lohmiller and Arnaud De Muyt contributed equally.     

My favourite molecule: Meiotin-1: The meiosis readiness factor?

Meiotin-1 is a protein found in developing microsporocytes of Lilium longiflorum, and immunological assays indicate that cognates exist in both mono- and dicotyledonous plants. Its temporal and spatial expression pattern, coupled with its unusual distribution in chromatin and the properties it shares with histone H1, encourages speculation that it is involved in regulating meiotic chromatin structure. Molecular analyses provide support for the hypothesis that meiotin-1 arose from histone H1 by an exon shuffling mechanism, as meiotin-1 is an H1-like protein that lacks the amino-terminal domain shared by H1 molecules. We have proposed that meiotin-1 serves to limit chromatin condensation in order to foster the unique cytological and molecular events which occur during meiotic prophase. As such, meiotin-1 fits the role of a ‘meiosis readiness factor’, and its accumulation to a threshold level may commit mitotically dividing progenitor cells to differentiate into meiocytes.     

Temporal and spatial distribution of meiotin-1 in anthers of Lilium longiflorum

Meiotin-1 is a chromatin associated, conserved protein found in meiocytes immediately preceding and during meiosis and is thought to have a role in determining the higher order structure of meiotic chromosomes [Riggs and Hasenkampf: Chromosoma 101:92–98, 1991]. In the studies reported here we utilized immunoblotting and immunocytochemical techniques to examine the temporal and spatial distribution of meiotin-1 in the anthers of Lilium longiflorum. The results with the anti-meiotin-1 immune serum were compared with those obtained using an anti-his-tone Hl immune serum. The anti-histone Hl immune serum gave constant immunostaining in all cell types of the anther at all of the stages tested. In contrast, the anti-meiotin-1 immune serum only gave immunostaining with the microsporocytes and to a lesser extent with the nutritive layer, the tapetum. It did not react with the cells of the anther wall. Meiotin-1 immunostaining was first present in significant quantities in the microsporocytes as they accumulated in the G1 phase before the onset of premeiotic S phase and reached peak levels in the time interval between leptotene and pachytene—the same interval when chromosome synapsis occurs and when reciprocal genetic exchange is thought to occur. Immunostaining for both meiotin-1 and histone H1 uniformly decorates the longitudinal axes of the chromosomes. Our data are consistent with the idea that the role of meiotin-1 may be to tag certain sequences or to limit the degree of chromosome condensation that occurs during meiotic prophase. © 1993 Wiley-Liss, Inc.     

Ameiotic, a gene that controls meiotic chromosome and cytoskeletal behavior in maize.

Microtubule organization during the novel cell division of ameiotic microsporocytes was examined using indirect immunofluorescence microscopy. A recessive mutation of the maize gene Ameiotic causes the replacement of meiosis I with a synchronized mitotic division (Palmer, R. G. (1971). Chromosoma 35, 233-246). All identifiable cytological features of this division, including chromosome behavior and microtubule organization, were typical of somatic cell division. Significantly, a cortical microtubule band was observed during prophase in ameiotic cells. In most somatic plant cells, a preprophase band of microtubules (PPB) predicts the cortical site where the future cell plate will join the sidewall. Similar structures, however, are absent in all meiotic and postmeiotic reproductive cells examined to date. These disruptions are consistent with a model where the wild-type Ameiotic gene encodes a product which acts during or before G2 and is necessary for initiating several independent meiotic processes, including both meiotic chromosome behavior and microtubule organization. The ameiotic mutation provides additional evidence that aspects of cytoskeletal organization unique to meiosis are genetically controlled. Finally, the presence of a PPB during the ameiotic division supports a model whereby multiple mechanisms are used to determine and maintain division plane polarity during normal meiosis.     

Meiosis readiness in Lilium longiflorum “Croft”

It is proposed that anthers of Lilium longiflorum Croft approaching the end of premeiotic mitosis reach a state described as meiosis readiness after which cells in premeiotic prophase are unable to complete a mitotic division but despiralize to interphase and enter a meiotic division. Many of the laggard premeiotic cells begin despiralization before reaching an extremely contracted state of late prophase. Premeiotic despiralization is not, therefore, attributed to a deficiency in metaphase but to an inability of these cells to complete prophase. Premeiotic despiralization appears to be preceded by a slowing-down of prophase development. There is variation among anthers and anther regions in the onset of prophase retardation and meiosis readiness. It is suggested that meiosis readiness depends upon a gradual accumulation of meiosis-inducing substances in the cytoplasm of the premeiotic cells. It has not been determined whether the cells that undergo premeiotic despiralization give rise to the giant microsporocytes with shattered chromosomes observed at late prophase of meiosis.     

A cytological approach to studying meiotic recombination and chromosome dynamics in Arabidopsis thaliana male meiocytes in three dimensions

During meiotic prophase I chromosomes undergo dramatic conformational changes that accompany chromosome condensation, pairing and recombination between homologs. These changes include the anchoring of telomeres to the nuclear envelope and their clustering to form a bouquet. In plants, these events have been studied and illustrated in intact meiocytes of species with large genomes. Arabidopsis thaliana is an excellent genetic model in which major molecular pathways that control synapsis and recombination between homologs have been uncovered. Yet the study of chromosome dynamics is hampered by current cytological methods that disrupt the three‐dimensional (3D) architecture of the nucleus. Here we set up a protocol to preserve the 3D configuration of A. thaliana meiocytes. We showed that this technique is compatible with the use of a variety of antibodies that label structural and recombination proteins and were able to highlight the presence of clustered synapsis initiation centers at the nuclear periphery. By using fluorescence in situ hybridization we also studied the behavior of chromosomes during pre‐meiotic G₂ and prophase I, revealing the existence of a telomere bouquet during A. thaliana male meiosis. In addition we showed that the number of telomeres in a bouquet and its volume vary greatly, thus revealing the complexity of telomere behavior during meiotic prophase I. Finally, by using probes that label subtelomeric regions of individual chromosomes, we revealed differential localization behaviors of chromosome ends. Our protocol opens new areas of research for investigating chromosome dynamics in A. thaliana meiocytes.     

HAPLOIDY IN HAPLOPAPPUS GRACILIS (N = 2)

Analysis of meiosis in a haploid (monoploid) sporophyte of Haplopappus gracilis (n = 2) showed a nonrandom distribution of chromosomes at anaphase I. Chromosomes A and B were associated at various prophase I stages in 34 % of the microsporocytes. Presumably, this association persisted long enough to disrupt random distribution in a portion of those meiocytes showing such an association. Pollen stainability of 26.5 % in the haploid was in agreement with the normal expectation. However, this number resulted from a nonrandom chromosome distribution. The usual method of predicting fertility of haploids, 1/2ⁿ, is not accurate for monoploids with low chromosome numbers, and more functional methods are proposed.     

The Caenorhabditis elegans SCC-3 homologue is required for meiotic synapsis and for proper chromosome disjunction in mitosis and meiosis

The product of the Caenorhabditis elegans ORF F18E2.3 is homologous to the cohesin component Scc3p. By antibody staining the product of F18E2.3 is found in interphase and early meiotic nuclei. At pachytene it localizes to the axes of meiotic chromosomes but is no longer detectable on chromatin later in meiosis or in mitoses. Depletion of the gene product by RNAi results in aberrant mitoses and meioses. In meiosis, homologous pairing is defective during early meiotic prophase and at diakinesis there occur univalents consisting of loosely connected sister chromatids or completely separated sisters. The recombination protein RAD-51 accumulates in nuclear foci at higher numbers during meiotic prophase and disappears later than in wild-type worms, suggesting a defect in the repair of meiotic double-stranded DNA breaks. Embryos showing nuclei of variable size and anaphase bridges, indicative of mitotic segregation defects, are frequently observed. In the most severely affected gonads, nuclear morphology cannot be related to any specific stage. The cytological localization and the consequences of the lack of the protein indicate that C. elegans SCC-3 is essential for sister chromatid cohesion both in mitosis and in meiosis.     

DNA-DEPENDENT FORMATION OF THE SYNAPTINEMAL COMPLEX AT MEIOTIC PROPHASE

Evalaution of microsporocytes cultured during discrete periods of meiotic prophase in the presence of deoxyadenosine, an inhibitor of DNA synthesis, indicate that: (1) late leptonema or early zygonema DNA synthesis is required to initiate the formation of the synaptinemal complex; (2) DNA synthesized during late zygonema is necessary for the disjunction of the paired homologs at diplonema; and (3) DNA synthesis in pachynema is a requisite for normal anaphase II separation of sister chromatids.