Sister chromatid separation and monopolar spindle organization in the first meiosis as two mechanisms of unreduced gametes formation in wheat-rye hybrids.

KEY MESSAGE: Unreduced gametes. The absence of a strict pachytene checkpoint in plants presents an opportunity to study meiosis in polyhaploid organisms. In the present study, we demonstrate that meiosis is coordinated in hybrids between disomic wheat-rye substitution lines 1Rv(1A), 2R(2D), 5R(5D), 6R(6A) and rye (Triticum aestivum L. × Secale cereale L., 4x = 28, ABDR). By using in situ hybridization with a centromere pAet6-09 probe and immunostaining with H3Ser10ph-, CENH3-, and α-tubulin-specific antibodies, we distinguished four chromosome behaviour types. The first one is a mitotic-like division that is characterized by mitotic centromere architecture, robust bipolar spindle, one-step loss of arm and centromere cohesion, and sister chromatid separation in the first and only meiotic division. The second type involves a monopolar spindle formation, which appears as a hat-shaped group of chromosomes moving in one direction, wherein MT bundles are co-oriented polewards. It prevents chromosome segregation in meiosis I, with a bipolar spindle distributing sister chromatids to the poles in meiosis II. These events subsequently result in the formation of unreduced microspores. The other two meiotic-like chromosome segregation patterns known as reductional and equational plus reductional represent stand-alone types of cell division rather than intermediate steps of meiosis I. Only sterile pollen is produced as a result of such meiotic-like chromosome behaviours. Slightly variable meiotic phenotypes are reproducibly observed in hybrids under different growth conditions. The 2R(2D)xR genotype tends to promote reductional division. In contrast, the genotypes 1Rv(1A)xR, 5R(5D)xR, and 6R(6A)xR promote equational chromosome segregation and monopolar spindle formation in addition to reductional and equational plus reductional division types.

Loading of the centromeric histone H3 variant during meiosis-how does it differ from mitosis?

In eukaryotic phyla studied so far, the essential centromeric histone H3 variant (CENH3) is loaded to centromeric nucleosomes after S-phase (except for yeast) but before mitotic segregation (except for metazoan). While the C-terminal part of CENH3 seems to be sufficient for mitotic centromere function in plants, meiotic centromeres neither load nor tolerate impaired CENH3 molecules. However, details about CENH3 deposition in meiocytes are unknown (except for Drosophila). Therefore, we quantified fluorescence signals after the immunostaining of CENH3 along meiotic and mitotic nuclear division cycles of rye, a monocotyledonous plant. One peak of fluorescence intensity appeared in the early meiotic prophase of pollen mother cells and a second one during interkinesis, both followed by a decrease of CENH3. Then, the next loading occurred in the male gametophyte before its first mitotic division. These data indicate that CENH3 loading differs between mitotic and meiotic nuclei. Contrary to the situation in mitotic cycles, CENH3 deposition is biphasic during meiosis and apparently linked with a quality check, a removal of impaired CENH3 molecules, and a general loss of CENH3 after each loading phase. These steps ensure an endowment of centromeres with a sufficient amount of correct CENH3 molecules as a prerequisite for centromere maintenance during mitotic cycles of the microgametophyte and the progeny. From a comparison with data available for Drosophila, we hypothesise that the post-divisional mitotic CENH3 loading in metazoans is evolutionarily derived from the post-divisional meiotic loading phase, while the pre-divisional first meiotic loading has been conserved among eukaryotes.

Abnormal mitosis induced by wheat-rye 1R monosomic addition lines.

Octoploid triticale were derived from common wheat (Triticum aestivum L. 'Mianyang11') × rye (Secale cereale L. 'Kustro'), and some progeny were obtained by the backcrossing of triticale with 'Mianyang11' followed by self-fertilization. In situ hybridization using rye genomic DNA and repetitive sequences pAs1 and pSc119.2 as probes was used to analyze the mitotic chromosomes of these progeny. Three wheat-rye 1R monosomic addition lines and a wheat line (12FT-1685) containing a 1R and a 1BL.1RS translocation chromosome were identified. Abnormal mitosis was observed in the two lines. During mitosis of a 1R monosomic addition line (3-8-20-1R-2), lagging chromosomes, micronuclei, chromosomal bridges, and the one pole segregation of 1R chromosome were observed. Abnormal mitotic behaviour of chromosomes was also observed in some of the self-progeny plants of lines 12FT-1685 and 3-8-20-1R-2. These progeny contained 1R chromosome or 1R chromosome arm. In addition, 4B chromosomes were absent from one of the progeny of 3-8-20-1R-2. This abnormal mitotic behaviour of chromosomes was not observed in two other 1R monosomic addition lines. These results indicate that a single 1R chromosome added to wheat might cause abnormal mitotic behaviour of both wheat and rye chromosomes and different genetic variations might occurr among the sibling 1R monosomic addition lines.

Dynamics of rye chromosome 1R regions with high or low crossover frequency in homology search and synapsis development.

In many organisms, homologous pairing and synapsis depend on the meiotic recombination machinery that repairs double-strand DNA breaks (DSBs) produced at the onset of meiosis. The culmination of recombination via crossover gives rise to chiasmata, which locate distally in many plant species such as rye, Secale cereale. Although, synapsis initiates close to the chromosome ends, a direct effect of regions with high crossover frequency on partner identification and synapsis initiation has not been demonstrated. Here, we analyze the dynamics of distal and proximal regions of a rye chromosome introgressed into wheat to define their role on meiotic homology search and synapsis. We have used lines with a pair of two-armed chromosome 1R of rye, or a pair of telocentrics of its long arm (1RL), which were homozygous for the standard 1RL structure, homozygous for an inversion of 1RL that changes chiasma location from distal to proximal, or heterozygous for the inversion. Physical mapping of recombination produced in the ditelocentric heterozygote (1RL/1RL(inv)) showed that 70% of crossovers in the arm were confined to a terminal segment representing 10% of the 1RL length. The dynamics of the arms 1RL and 1RL(inv) during zygotene demonstrates that crossover-rich regions are more active in recognizing the homologous partner and developing synapsis than crossover-poor regions. When the crossover-rich regions are positioned in the vicinity of chromosome ends, their association is facilitated by telomere clustering; when they are positioned centrally in one of the two-armed chromosomes and distally in the homolog, their association is probably derived from chromosome elongation. On the other hand, chromosome movements that disassemble the bouquet may facilitate chromosome pairing correction by dissolution of improper chromosome associations. Taken together, these data support that repair of DSBs via crossover is essential in both the search of the homologous partner and consolidation of homologous synapsis.

Flow cytometric chromosome sorting in plants: the next generation.

Genome analysis in many plant species is hampered by large genome size and by sequence redundancy due to the presence of repetitive DNA and polyploidy. One solution is to reduce the sample complexity by dissecting the genomes to single chromosomes. This can be realized by flow cytometric sorting, which enables purification of chromosomes in large numbers. Coupling the chromosome sorting technology with next generation sequencing provides a targeted and cost effective way to tackle complex genomes. The methods outlined in this article describe a procedure for preparation of chromosomal DNA suitable for next-generation sequencing.

Unequal chromosome division and inter-genomic translocation occurred in somatic cells of wheat-rye allopolyploid.

Newly synthesized wheat-rye allopolyploids were investigated by genomic in situ hybridization, over the first, second, third and fourth allopolyploid generations. Inter and intra chromosome connections were observed in 12 root-tip cells of CA4.4.7 (S(2) generation), and translocations between wheat and rye chromosomes were also detected in five root-tip cells. In root-tip cells of CA4.4.7.5 and CA4.4.7.2.2 (S(3) and S(4) generation), the chromosome connections occurred again, a dissociative small rye segment was detected in seven cells of CA4.4.7.5. In plants MSV6.1 and MSV6.5 (S(1) generation), almost half of the root-tip cells contained 13 rye chromosomes and the rest held 12 rye chromosomes, and all the cells of the two plants contained 42 wheat chromosomes. Five pairing configurations of rye chromosomes, including 5 II + 3 I, 6 II + 1 I, 6 II, 5 II + 2 I and 4 II + 4 I, were observed in pollen mother cells of the two plants. The two plants' progeny, including S(2), S(3), and S(4) generation plants, contained 42 wheat chromosomes and 12 rye chromosomes. Therefore, the inter chromosome translocation and unequal chromosome division could occur in somatic cells of wide hybrids. The unequal chromosome division in somatic cell could induce chromosome elimination at the early stages of allopolyploidization.

Activation of rye 5RL neocentromere by an organophosphate pesticide.

An interstitial constriction located on the long arm of rye chromosome 5R (5RL) shows neocentromeric activity at meiosis. In some meiocytes this region is strongly stretched orienting with the true centromere to opposite poles at metaphase I, and keeping sister chromatid cohesion at anaphase I. We found previously that the frequency of neocentric activity varied dramatically in different generations suggesting the effect of environmental factors. Here we studied the behavior of the 5RL neocentromere in mono- and ditelosomic 5RL, and mono-, and disomic 5R wheat-rye addition lines, untreated and treated with an organophosphate pesticide. The treated plants form neocentromeres with an about 4.5-fold increased frequency compared to untreated ones, demonstrating that the pesticide promotes neocentric activity. The neocentromere was activated irrespectively of the pairing configuration or the presence of a complete or truncated 5R centromere. Fluorescence in situ hybridization (FISH) with 2 repetitive sequences (UCM600 and pSc119.2) present at the constriction showed kinetic activity at several locations within this region. Immunostaining with anti-α-tubulin showed that treated plants have abnormal spindles in 46% of the metaphase I cells, indicating that disturbances in spindle formation might promote neocentromere activation.

Chiasma frequency is region specific and chromosome conformation dependent in a rye chromosome added to wheat.

In many plant species synapsis starts at, or close to, the chromosome ends and this has been considered to be related to the distal location of chiasmata. In this regard we have studied the meiotic behavior of rye chromosome pair 5R in a wheat background using fluorescence in situ hybridization. The use of different DNA probes allowed the identification of the 2 rye homologues, their centromeres and subtelomeric heterochromatic chromomeres, and the telomeres of all chromosomes in prophase I and metaphase I. Three types of plants were analyzed: homozygotes for the standard chromosome 5R, homozygotes for a deficient chromosome 5R (del5R) with only the proximal 30% of its long arm (del5RL) and heterozygotes. Synapsis of the deficient chromosome arm pair del5RL was completed in most meiocytes at pachytene but the number of chiasmata formed was much lower than in the intact 5RL arm. Deletion facilitated the migration of the telomere of the accompanying chromosome arm 5RS during bouquet organization. This was followed by an increase of synapsis and chiasma frequency in this arm with regard to its counterpart of the intact chromosome. Results demonstrate that crossover formation depends on the DNA sequence or the chromatin organization of each chromosome region and that homologous alignment, synapsis and chiasma formation may be conditioned by chromosome conformation.

The Ph1 locus from wheat controls meiotic chromosome pairing in autotetraploid rye (Secale cereale L.).

The Ph1 locus on chromosome 5B enforces strictly bivalent pairing in polyploid wheat, but the exact mechanism of its action remains unknown. Pairing restriction involves not only wheat homoeologues and all alien introgressions but also differentiated homologues. In this study we show that chromosome 5B with its Ph1 locus also controls chromosome pairing in autotetraploid rye by apparently restricting chiasma formation between dissimilar homologues. Unlike in wheat, the effect appears to be dosage-dependent, which may be a reflection of an interaction between Ph1 and the rye chromosome pairing control system. With 2 doses of Ph1 present, chiasmate pairing was severely restricted resulting in a significantly higher number of univalents and bivalents per cell than in the controls. The restrictions imposed by Ph1 virtually eliminated MI pairing of chromosome arms polymorphic for their C-band patterns and did not appear to affect arms with similar patterns. If the polymorphism for C-bands is taken as a measure of the overall chromosome similarity/divergence, such differences were recognized and acted upon by the Ph1 locus. The fact that Ph1 operates in rye in the same fashion as in polyploid wheats suggests that it controls some basic mechanism of chromosome recognition.

Structural characterization of feruloylated arabinoxylans and xylans released from water-unextractable cell walls of rye outer layers upon treatment with lichenase and cellulase.

Destarched and deproteinated water-unextractable material (WUM) of rye outer layers was sequentially treated with lichenase and cellulase to digest beta-glucans and a part of the cellulose. As a result, the polymeric cell-wall material (CWM) initially associated with these polysaccharides was released into solution (AXL and AXC for lichenase- and cellulase-extractable fractions, respectively). A portion of the material that self-aggregated during extractions was further solubilized with DMSO (XD and XD-P for the fraction left in the solution and that precipitated during dialysis, respectively). Arabinoxylans (AXs) recovered from these fractions were composed of populations with different degrees of substitution with alpha-l-arabinofuranosyl residues (Araf). Their counterparts present in the AXL and AXC fractions exhibited higher (0.60 and 0.75) arabinose-to-xylose ratios (Ara/Xyl) and represented 27% and 32% of the isolated AXs, respectively. The xylans of the XD and XD-P fractions had a very low Ara/Xyl ratio (0.16 and 0.09) and accounted for 23% and 18%, respectively. Based on the results of ammonium sulfate fractionation and sugar analysis, it has been shown that AXL consisted of AX subfractions having Ara/Xyl in a narrow range (0.50-0.66). By contrast, the cellulase-extractable AXs were characterized by the presence of the highly branched subfractions (Ara/Xyl of 1.00) as well. Quite unexpectedly, the higher amounts of ferulic acid (FA) were found in the cell-wall fractions enriched in xylans than in the AX-containing fractions. Furthermore, as demonstrated by (1)H NMR and Fourier transform infrared spectroscopy, xylans were substituted with alpha-d-glucuronopyranosyl residues (GlcpA).

[Additional phragmoplast corrects abnormal cytokinesis in wheat x rye hybrid pollen mother cells].

The phragmoplast dysfunction in wheat x rye hybrid F1 male meiosis has been described. The pollen mother cells (PMCs) show the phenotype where transition from central spindle fibers (forming a solid bundle) to a phragmoplast (hollow cylinder) is blocked. The blockade suppresses centrifugal movement of the phragmoplast and cell plate formation. The resulting cells occur to be binucleate. Sometimes, the two nuclei join and form one restitution nucleus. PMCs of wheat x rye F1 hybrid N D-144gp 06r. F1 (T. aestivum c. 93-60 T 9 x S. cereale c. Saratovskaya 7) showing this phenotype have an additional phragmoplast at late telophase. This happens like that in the case of immobile phragmoplast formation in meiosis in bicotyledons: the new phragmoplast arises by the aid of microtubules polymerization starting from the spindle poles. The new additional phragmoplast builds a new cell plate and accomplishes cytokinesis.

Discrimination of repetitive sequences polymorphism in Secale cereale by genomic in situ hybridization-banding.

Genomic in situ hybridization banding (GISH-banding), a technique slightly modified from conventional GISH, was used to probe the Chinese native rye (Secale cereale L.) DNA, and enabled us to visualize the individual rye chromosomes and create a universal reference karyotype of the S. cereale chromosome 1R to 7R. The GISH-banding approach used in the present study was able to discriminate S. cereale chromosomes or segments in the wheat (Triticum aestivum L.) background, including the Triticale, wheat-rye addition and translocation lines. Moreover, the GISH-banding pattern of S. cereale subsp. Afghanicum chromosomes was consistent with that of Chinese native rye cv. Jingzhou rye; whereas the GISH-banding pattern of Secale vavilovii was different from that of S. cereale, indicating that GISH-banding can be used to study evolutionary polymorphism in species or subspecies of Secale. In addition, the production and application of GISH-banding to the study of adenine-thymine-riched heterochromatin is discussed.

Effects on genome constitution and novel cell wall formation caused by the addition of 5RS rye chromosome to common wheat.

The cytological instability of common wheat-rye addition lines was investigated in the present study. The chromosome numbers of almost all addition lines were considerably stable, but those of CS + 5R were very variable. The rye chromosome added in this line was found to be much shorter than expected. Fluorescent in situ hybridization with 5S rDNA and the centromere-specific probes clearly revealed that the short rye chromosome contains only a short arm of chromosome 5R (5RS). In this line, chromosome numbers of both 5RS and common wheat were changeable. The chromosome numbers ranged from 2n = 36 to 2n = 44 in the cells carrying two 5RS, and ranged from 2n = 31 to 2n = 44 in one 5RS cells. In addition to the chromosome instability, the multicells wrapped in a sac-like structure were frequently observed in the root meristematic tissues of CS + 5RS after the enzyme treatment for chromosome preparation. Genomic in situ hybridization with rye DNA as a probe showed that all cells in sacs investigated were at the interphase stage and contained one or two 5RS chromosomes. An electron microscopic analysis revealed that the cells of CS + 5RS, particularly in sacs, have abnormal (irregular and curved) cell walls. These results indicate that 5RS has (a) specific factor(s) influencing the cell wall development as well as the genome stability.

Meiotic genes and proteins in cereals.

We review the current status of our understanding and knowledge of the genes and proteins controlling meiosis in five major cereals, rye, wheat, barley, rice and maize. For each crop, we describe the genetic and genomic infrastructure available to investigators, before considering the inventory of genes and proteins that have roles to play in this process. Emphasis is given throughout as to how translational genomic and proteomic approaches have enabled us to circumvent some of the intractable features of this important group of plants.

Nuclear architecture and chromosome dynamics in the search of the pairing partner in meiosis in plants.

The formation of haploid gametes in organisms with sexual reproduction requires regular bivalent chromosome pairing in meiosis. In many species, homologous chromosomes occupy separate territories at the onset of meiosis. To be paired at metaphase I, they need to be brought into a close proximity for interactions that include homology recognition and the establishment of some form of bonds. How homologues find each other is one of the least understood meiotic events. Plant species with large or medium sized genomes, such as wheat or maize, are excellent materials for the cytological analysis of chromosome dynamics at early meiosis, but genes that control meiosis have been identified mainly in small genome species such as Arabidopsis thaliana. This review is focused on the contribution studies on plants are providing to the knowledge of the initial steps of the meiotic process.

[Genetic regulation of the centromere division in rye and wheat univalent chromosomes in dimonosomics during meiotic anaphase I].

Cytogenetic analysis of meiosis in the wheat--rye dimonosomics 1Rv-1A, 1Ron-1A, 2R-2D, 5R-5A, and 6R-6A was conducted. C-banding was used to study the segregation pattern of each of two univalent chromosomes during the first meiotic division. It has been shown that the division frequency of the centromeric regions of all rye chromosomes in the pair studied is significantly higher than in the wheat chromosomes. The ANOVA performed suggest that the plant genotype contributes significantly (at P = 0.05) to the behavior pattern of univalent chromosomes in meiosis. The data obtained demonstrate that the rye and wheat chromosomes studied are involved in genetic regulation of centromere division in meiotic anaphase I (AI). The presence of rye chromosome 2R and wheat chromosome 2D suppresses the division of centromeres of the sister chromatids in AI. Rye chromosomes 1Rv, 1Ron, 5R, and 6R induce equational division; however, rye chromosome 1Rv increases to a greater degree the frequency of equational division of wheat chromosome 1A as compared with chromosome 1Ron.

Epigenetic control may explain large within-plant heterogeneity of meiotic behavior in telocentric trisomics of rye.

In telocentric trisomics (telotrisomics) of organisms in which the chromosomes normally have two distinct arms, a single chromosome arm with a centromere is present in addition to a complete diploid set of chromosomes. It is the simplest form of polysomy and suitable for analyzing meiotic pairing and recombination patterns in situations where chromosomes compete for pairing. When no suitable meiotic chromosome markers are available, four metaphase I configurations can be distinguished. Their relative frequencies are indicative of the pairing and recombination patterns. In short arm (1RS) telotrisomics of chromosome 1R of rye (Secale cereale) we observed great differences in pairing and recombination patterns among spikes from different tillers and clones of the same plants. Anthers within spikes were only very rarely different. We analyzed a large number of genotypes, including inbreds as well as hybrids. The effects of genetic and environmental conditions on heterogeneity, if any, were limited. Considering that the reproductive tissue of a spike is derived from one primordial cell, it seems that at the start of sexual differentiation there was variation among cells in chromosomal control, which at meiosis determines pairing and crossing-over competence. We suggest that it is an epigenetic system that rigidly maintains this pattern through generative differentiation. In competitive situations the combination most competent for pairing will pair preferentially, forming specific meiotic configurations with different frequencies for different spikes of the same plant. This would explain the heterogeneity between spikes and the homogeneity within spikes. The epigenetic system could involve chromatin conformation or DNA methylation. There were no signs of heterochromatinization.

Dissecting meiosis of rye using translational proteomics.

BACKGROUND AND AIMS: Much of our understanding of the genetic control of meiosis has come from recent studies of model organisms, which have given us valuable insights into processes such as recombination and the synapsis of chromosomes. The challenge now is to determine to what extent these models are representative of other groups of organisms, and to what extent generalisations can be made as to how meiosis works. Through a comparative proteomic approach with Arabidopsis thaliana, this study describes the spatial and temporal expression of key structural and recombinogenic proteins of cereal rye (Secale cereale). METHODS: Antibodies to two synaptonemal complex-associated proteins (Asy1 and Zyp1) and two recombination-related proteins (Spo11 and Rad51) of A. thaliana were bound to meiocytes throughout meiotic prophase of rye, and visualized using conventional fluorescence microscopy and confocal laser scanning microscopy. Western analysis was performed on proteins extracted from pooled prophase I anthers, as a prelude to more advanced proteomic investigations. KEY RESULTS: The four antibodies of A. thaliana reliably detected their epitopes in rye. The expression profile of Rad51 is consistent with its role in recombination. Asy1 protein is shown for the first time to cap the ends of bivalents. Western analysis reveals structural variants of the transverse filament protein Zyp1. CONCLUSIONS: Asy1 cores are assembled by elongation of early foci. The persistence of foci of Spo11 to late prophase does not fit the current model of molecular recombination. The putative structural variants of Zyp1 may indicate modification of the protein as bivalents are assembled.

Relationships between transcription, silver staining, and chromatin organization of nucleolar organizers in Secale cereale.

The nucleolar organizer regions (NORs) are composed of hundreds of rRNA genes, typically spanning several megabases. Cytologically, NORs include regions that are highly condensed and regions that are decondensed, the latter corresponding to regions at which associated proteins stain intensively with silver (Ag-NORs) and where active rRNA gene transcription is thought to occur. To test the relationship between rRNA gene activity, NOR silver staining, and rDNA (genes coding for rRNA) chromatin condensation, we used the DNA methyl-transferase inhibitor 5-azacytidine to evaluate the correlation between the epigenetic regulation of rRNA genes and NOR silver staining in the plant Secale cereale. Following 5-azacytidine treatment, we observed an increase in rRNA gene transcription as well as a reduction in the number of cells showing a significant difference in the size of the silver-stained domains in the two NORs. These transcriptional changes occurred concomitantly with an increase in nuclear and nucleolar size and were associated with the reallocation of most of the rDNA from perinucleolar heterochromatin into the nucleolus. Collectively, these results suggest that rRNA gene transcription, silver staining, and NOR decondensation are interrelated in S. cereale.

Transcriptionally active heterochromatin in rye B chromosomes.

B chromosomes (Bs) are dispensable components of the genomes of numerous species. Thus far, there is a lack of evidence for any transcripts of Bs in plants, with the exception of some rDNA sequences. Here, we show that the Giemsa banding-positive heterochromatic subterminal domain of rye (Secale cereale) Bs undergoes decondensation during interphase. Contrary to the heterochromatic regions of A chromosomes, this domain is simultaneously marked by trimethylated H3K4 and by trimethylated H3K27, an unusual combination of apparently conflicting histone modifications. Notably, both types of B-specific high copy repeat families (E3900 and D1100) of the subterminal domain are transcriptionally active, although with different tissue type-dependent activity. No small RNAs were detected specifically for the presence of Bs. The lack of any significant open reading frame and the highly heterogeneous size of mainly polyadenylated transcripts indicate that the noncoding RNA may function as structural or catalytic RNA.