Immunofluorescent synaptonemal complex analysis in azoospermic men

The molecular cause of germ cell meiotic defects in azoospermic men is rarely known. During meiotic prophase I, a proteinaceous structure called the synaptonemal complex (SC) appears along the pairing axis of homologous chromosomes and meiotic recombination takes place. Newly-developed immunofluorescence techniques for SC proteins (SCP1 and SCP3) and for a DNA mismatch repair protein (MLH1) present in late recombination nodules allow simultaneous analysis of synapsis, and of meiotic recombination, during the first meiotic prophase in spermatocytes. This immunofluorescent SC analysis enables accurate meiotic prophase substaging and the identification of asynaptic pachytene spermatocytes. Spermatogenic defects were examined in azoospermic men using immunofluorescent SC and MLH1 analysis. Five males with obstructive azoospermia, 18 males with nonobstructive azoospermia and 11 control males with normal spermatogenesis were recruited for the study. In males with obstructive azoospermia, the fidelity of chromosome pairing (determined by the percentage of cells with gaps [discontinuities]/splits [unpaired chromosome regions] in the SCs, and nonexchange SCs [bivalents with 0 MLH1 foci]) was similar to those in normal males. The recombination frequencies (determined by the mean number of MLH1 foci per cell at the pachytene stage) were significantly reduced in obstructive azoospermia compared to that in controls. In men with nonobstructive azoospermia, a marked heterogeneity in spermatogenesis was found: 45% had a complete absence of meiotic cells; 5% had germ cells arrested at the zygotene stage of meiotic prophase; the rest had impaired fidelity of chromosome synapsis and significantly reduced recombination in pachytene. In addition, significantly more cells were in the leptotene and zygotene meiotic prophase stages in nonobstructive azoospermic patients, compared to controls. Defects in chromosome pairing and decreased recombination during meiotic prophase may have led to spermatogenesis arrest and contributed in part to this unexplained infertility.     

Lateral elements inside synaptonemal complex-like polycomplexes in ndt80 mutants of yeast bind DNA

The synaptonemal complex (SC) keeps the synapsed homologous chromosomes together during pachytene in meiotic prophase I. Structures that resemble stacks of SCs, polycomplexes, are sometimes found before or after pachytene. We have investigated ndt80 mutants of yeast, which arrest in pachytene. SCs appear normal in spread chromosome preparations, but are only occasionally found in intact nuclei examined in the electron microscope. Instead, large polycomplexes occur in almost every ndt80 mutant nucleus. Immunoelectron microscopy using DNA antibodies show strong preferential labeling to the lateral element parts of the polycomplexes. In situ hybridization using chromosome-specific probes confirms that the chromosomes in ndt80 mutants are paired and attached to the SCs. Our results suggest that polycomplexes can be involved in binding of chromosomes and possibly also in synapsis.     

CENH3 morphogenesis reveals dynamic centromere associations during synaptonemal complex formation and the progression through male meiosis in hexaploid wheat

During meiosis, centromeres in some species undergo a series of associations, but the processes and progression to homologous pairing is still a matter of debate. Here, we aimed to correlate meiotic centromere dynamics and early telomere behaviour to the progression of synaptonemal complex (SC) construction in hexaploid wheat (2n = 42) by triple immunolabelling of CENH3 protein marking functional centromeres, and SC proteins ASY1 (unpaired lateral elements) and ZYP1 (central elements in synapsed chromosomes). We show that single or multiple centromere associations formed in meiotic interphase undergo a progressive polarization (clustering) at the nuclear periphery in early leptotene, leading to formation of the telomere bouquet. Critically, immunolabelling shows the dynamics of these presynaptic centromere associations and a structural reorganization of the centromeric chromatin coinciding with key events of synapsis initiation from the subtelomeric regions. As short stretches of subtelomeric synapsis emerged at early zygotene, centromere clusters lost their strong polarization, gradually resolving as individual centromeres indicated by more than 21 CENH3 foci associated with unpaired lateral elements. Only following this centromere depolarization were homologous chromosome arms connected, as observed by the alignment and fusion of interstitial ZYP1 loci elongating at zygotene so synapsis at centromeres is a continuation of the interstitial synapsis. Our results thus reveal that centromere associations are a component of the timing and progression of chromosome synapsis, and the gradual release of the individual centromeres from the clusters correlates with the elongation of interstitial synapsis between the corresponding homologues.     

Synaptonemal complexes in insects

The synaptonemal complex (SC) is the key nuclear element formed in meiotic prophase I to join 2 homologous chromosomes at the pachytene bivalent. It is a highly conserved structure that is universally present in eukaryotes. The SC is presented as a tripartite protein structure, which consists of 2 lateral elements and a central region. In insects, the central region is particularly distinct and highly ordered. This made it possible to describe the fine structure of the central region and propose a model of its architecture. Chromatid DNA is arranged in chromatin loops extending radially from the SC. The loops appear to consist of a basic chromatin fiber with a diameter of 20–30 nm. In many insect species, synaptonemal polycomplexes occur in postpachytene cells. They represent one of the possible ways of SC degradation. Another process, which occurs beyond pachytene, is the formation of proteinaceous chromatid axis, the silver-stained chromatid core. Based on results in insect models, the chromatid cores have been related to the structure and formation of the SC. Research on insect models significantly contributed to understanding individual steps of the SC formation and temporal sequence of chromosome pairing. These include the formation of lateral elements of the SC, pairing initiation, interlocking of chromosomes, and synapsis of homologous chromosomes. Attention is also given to non-homologous pairing, including synaptic adjustment, correction of pairing, and pairing of sex chromosomes. In the next section, chiasmatic and achiasmatic modes of meiosis are compared with respect to the SC formation. In the chiasmatic mode, the SCs display recombination nodules that are believed to mediate the process of recombination. These nodules were discovered in insects, and indirect evidence for their role comes from insects. Two different examples of achiasmatic meiosis, occurring in the heterogametic sex of several insect orders, are given: one involves the SC formation, whereas in the other, SCs are absent. Finally, the potential of SC karyotyping for analysis of the insect genome is discussed.     

Meiotic mutants of rye Secale cereale L. II. The nonhomologous synapsis in desynaptic mutants sy7 and sy10

We studied the expression and inheritance of two spontaneous mutations found in different populations of rye Secale cereale L. that cause high univalent frequency in meiosis and low fertility. Both mutations were inherited as monogenic recessives. For each of the mutations the corresponding gene symbols (sy7 and sy10) were suggested although their allelism has not been studied. These mutants differ in chiasma frequency and in the number of univalents per meiocyte. Electron microscopy of the wholemount surface-spread synaptonemal complexes (SCs) from microsporocytes of both mutants revealed that during meiotic prophase I random synapsis began and progressed that involved not only homologous but also nonhomologous chromosomes. SCs were formed with frequent changes of pairing partners (switches) and intrachromosomal foldbacks of unpaired axial elements. As a result, incompletely synapsed, non-homologous and multivalent SCs were formed in mutants by the stage analogous to pachytene in normal plants. In sy7 a maximum in the number of switches and foldbacks were observed at zygotene, whereas in sy10 this occurred at pachytene. We suggest that it is the process of recognition of homology that is impaired in both mutants. This leads to indiscriminate synapsis and prevents chiasma formation. Both mutants may be classified as desynaptic.     

The desynaptic (dy) and desynaptic1 (dsy1) mutations in maize (Zea mays L) cause distinct telomere-misplacement phenotypes during meiotic prophase

During meiotic prophase, telomeres actively attach themselves to the nuclear envelope and cluster in an arrangement called the bouquet. The bouquet is unique to meiosis, highly conserved, and thought to facilitate homologous chromosome synapsis. Analy sis of three-dimensional fluorescence in situ hybridization (3-D FISH) image data has been employed to characterize the bouquet in fixed pollen mother cells of maize (Zea mays L.). In order to examine the function of the bouquet further, several meiotic mutants were screened for telomeric defects using 3-D FISH as an assay. Two mutants, desynaptic (dy) and desynaptic1 (dsy1), were found to exhibit novel telomere-misplacement phenotypes. In both cases, the telomere-associated mutant phenotypes occurred prior to what was previously reported as the earliest affected stage. Three alleles of the desynaptic1 mutation (dsy1-1, dsy1-9101, and dsy1-9307) resulted in a partial bouquet phenotype at the zygotene stage of meiotic prophase. By contrast, dy nuclei contained apparently normal bouquets, but then resulted in a premature intranuclear localization of telomeres at the pachytene stage, when telomeres normally disperse but remain attached to the nuclear envelope. The dsy1 mutation is known to impair the fidelity and progression of homologous synapsis, whereas the dy mutation is known to reduce recombination rates. If the telomere misplacements are primary defects of these mutants, then these data would be consistent with the hypothesis that meiotic telomeres have at least two separable functions, one involving proper homologous chromosome synapsis at the bouquet stage and another involving post-bouquet cross-over control.     

Expression and functional analysis of <Emphasis Type="Italic">TaASY1</Emphasis> during meiosis of bread wheat (<Emphasis Type="Italic">Triticum aestivum</Emphasis>)

Background  

Pairing and synapsis of homologous chromosomes is required for normal chromosome segregation and the exchange of genetic material via recombination during meiosis. Synapsis is complete at pachytene following the formation of a tri-partite proteinaceous structure known as the synaptonemal complex (SC). In yeast, HOP1 is essential for formation of the SC, and localises along chromosome axes during prophase I. Homologues in Arabidopsis (AtASY1), Brassica (BoASY1) and rice (OsPAIR2) have been isolated through analysis of mutants that display decreased fertility due to severely reduced synapsis of homologous chromosomes. Analysis of these genes has indicated that they play a similar role to HOP1 in pairing and formation of the SC through localisation to axial/lateral elements of the SC.     

Synaptonemal complex antigen location and conservation

The axial cores of chromosomes in the meiotic prophase nuclei of most sexually reproducing organisms play a pivotal role in the arrangement of chromatin, in the synapsis of homologous chromosomes, in the process of genetic recombination, and in the disjunction of chromosomes. We report an immunogold analysis of the axial cores and the synaptonemal complexes (SC) using two mouse monoclonal antibodies raised against isolated rat SCs. In Western blots of purified SCs, antibody II52F10 recognizes a 30- and a 33-kD peptide (Heyting, C., P. B. Moens, W. van Raamsdonk, A. J. J. Dietrich, A. C. G. Vink, and E. J. W. Redeker, 1987, Eur. J. Cell Biol., 43: 148-154). In spreads of rat spermatocyte nuclei it produces gold grains over the cores of autosomal and sex chromosomes. The cores label lightly during the chromosome pairing stage (zygotene) of early meiotic prophase and they become more intensely labeled when they are parallel aligned as the lateral elements of the SC during pachytene (55 grains/micron SC). Statistical analysis of electronically recorded gold grain positions shows that the two means of the bimodal gold grain distribution coincide with the centers of the lateral elements. At diplotene, when the cores separate, the antigen is still detected along the length of the core and the enlarged ends are heavily labeled. Shadow-cast SC preparations show that recombination nodules are not labeled. The continued presence suggests that the antigens serve a continuing function in the cores, such as chromatin binding, and/or structural integrity. Antibody III15B8, which does not recognize the 30- and 33-kD peptides, produces gold grains predominantly between the lateral elements. The grain distribution is bimodal with the mean of each peak just inside the pairing face of the lateral element. The antigen is present where and while the cores of the homologous chromosomes are paired. From the location and the timing, it is assumed that the antigen recognized by III15B8 functions in chromosome pairing at meiotic prophase. The two anti-rat SC antibodies label rat and mouse SCs but not rabbit or dog SCs. A positive control using human CREST (calcinosis, Raynaud's phenomenon, esophageal dysmotility, sclerodactyly, telangiectasia) anti-centromere serum gives equivalent labeling of SC centromeres in the rat, mouse, rabbit, and dog. It is concluded that the SC antigens recognized by II52F10 and III15B8 are not widely conserved. The two antibodies do not bind to cellular or nuclear components of somatic cells.(ABSTRACT TRUNCATED AT 400 WORDS)     

Differing requirements for RAD51 and DMC1 in meiotic pairing of centromeres and chromosome arms in Arabidopsis thaliana

During meiosis homologous chromosomes pair, recombine, and synapse, thus ensuring accurate chromosome segregation and the halving of ploidy necessary for gametogenesis. The processes permitting a chromosome to pair only with its homologue are not fully understood, but successful pairing of homologous chromosomes is tightly linked to recombination. In Arabidopsis thaliana, meiotic prophase of rad51, xrcc3, and rad51C mutants appears normal up to the zygotene/pachytene stage, after which the genome fragments, leading to sterility. To better understand the relationship between recombination and chromosome pairing, we have analysed meiotic chromosome pairing in these and in dmc1 mutant lines. Our data show a differing requirement for these proteins in pairing of centromeric regions and chromosome arms. No homologous pairing of mid-arm or distal regions was observed in rad51, xrcc3, and rad51C mutants. However, homologous centromeres do pair in these mutants and we show that this does depend upon recombination, principally on DMC1. This centromere pairing extends well beyond the heterochromatic centromere region and, surprisingly, does not require XRCC3 and RAD51C. In addition to clarifying and bringing the roles of centromeres in meiotic synapsis to the fore, this analysis thus separates the roles in meiotic synapsis of DMC1 and RAD51 and the meiotic RAD51 paralogs, XRCC3 and RAD51C, with respect to different chromosome domains.     

The maize desynaptic1 mutation disrupts meiotic chromosome synapsis

In most eukaryotes, homologous chromosomes undergo synapsis during the first meiotic prophase. A consequence of mutations that interfere with the fidelity or completeness of synapsis can be failure in the formation or maintenance of bivalents, resulting in univalent formation at diakinesis and production of unbalanced spores or gametes. Such mutations, termed desynaptic mutations, can result in complete or partial sterility. We have examined the effect of the maize desynaptic1-9101 mutation on synapsis, using the nuclear spread technique and electron microscopy to examine microsporocytes ranging from early pachytene until the diplotene stage of prophase I. Throughout the pachytene stage, there was an average of about 10 sites of lateral element divergence (indicating nonhomologous synapsis), and during middle and late pachytene, an average of two and three sites of foldback (intrachromosomal) synapsis, per mutant nucleus, respectively. By the diplotene stage, the number of sites of lateral element divergence had decreased to seven, and there was an average of one foldback synapsis site per nucleus. Lateral element divergence and foldback synapsis were not found in spread pachytene nuclei from normal plants. These results imply that the normal expression of the dsy1 gene is essential for the restriction of chromosome synapsis to homologues. The abundance of nonhomologous synapsis and the persistence of extended stretches of unsynapsed axial elements throughout the pachytene stage of dsy1–9101 meiocytes suggests that this mutation disrupts both the fidelity of homology search and the forward course of the synaptic process. This mutation may identify a maize mismatch repair gene. Dev. Genet. 21:146–159, 1997. © 1997 Wiley-Liss, Inc.     

Chromosome pairing in the allotetraploid Aegilops biuncialis and a triploid intergeneric hybrid.

Chromosome pairing behaviour of the natural allotetraploid Aegilops biuncialis (genome UUMM) and a triploid hybrid Ae. biuncialis x Secale cereale (genome UMR) was analyzed by electron microscopy in surface-spread prophase I nuclei. Synaptonemal-complex analysis at zygotene and pachytene revealed that synapsis in the allotetraploid was mostly between homologous chromosomes, although a few quadrivalents were also formed. Only homologous bivalents were observed at metaphase I. In contrast, homoeologous and heterologous chromosome associations were common at prophase I and metaphase I of the triploid hybrid. It is concluded that the mechanism controlling bivalent formation in Ae. biuncialis acts mainly at zygotene by restricting pairing to homologous chromosomes, but also acts at pachytene by preventing chiasma formation in the homoeologous associations. In the hybrid the mechanism fails at both stages. Key words : Aegilops biuncialis, allotetraploid, intergeneric hybrid, pairing control, synaptonemal complex.     

Identification of a structural protein component of rat synaptonemal complexes

Synaptonemal complexes (SCs) are evolutionarily conserved nuclear structures of meiotic cells which form during the zygotene stage of the first meiotic prophase and are responsible for the pairing of homologous chromosomes. Their formation appears to be a prerequisite for crossing-over events and proper chromosome segregation during the first meiotic division. Despite knowledge of their central role in genetic recombination processes very little is known about the molecular composition and the mechanisms governing the assembly of the SCs. In the present study we report on the characterization of a monoclonal antibody (SC14f10) which enabled us to identify a novel SC protein termed SC48. Protein SC48 has a Mr of 48,000 and migrates in two-dimensional gels with a pH value of 6.9. By means of immunogold EM we localized this protein to the central region of the SC. In cell fractionation experiments we recovered protein SC48 together with SC-residual structures in a karyoskeletal fraction of pachytene spermatocytes. Our results indicate that SC48 is a meiosis-specific structural protein component of the SC probably involved in the pairing of homologous chromosomes.     

Ultrastructure of meiotic pairing in B chromosomes of Crepis capillaris

The meiotic pairing behaviour of four B isochromosomes of Crepis capillaris was studied by synaptonemal complex (SC) surface spreading of pollen mother cells. The four B chromosomes form a tightly associated group, separate from the standard chromosomes, throughout zygotene and pachytene. All four B chromosomes are also folded around their axis of symmetry, the centromere, and the eight homologous arms are closely aligned from the earliest prophase I stages. A high frequency of multivalent pairing of the four B chromosomes is observed at pachytene, in excess of 90%, mirroring the situation observed at metaphase I but exceeding the frequency expected (76.2%) on the assumption of random pairing among the eight B isochromosome arms with a single distal pairing initiation site per arm. The higher than expected frequency of multivalents is due to the occurrence of multiple pairing initiations along the B isochromosome arms, resulting in high frequencies of pairing partner switches. Pairing of the standard chromosome set is frequently incomplete in the presence of four B chromosomes, and abnormalities of SC structure such as thickening and splitting of axes and lateral elements are also frequently seen. Similarly, B chromosomes show partial pairing failure, the extent of which is correlated with pairing failure in the standard chromosome set. The B chromosomes themselves also show abnormalities of SC structure. Both standard and B chromosomes show non-homologous foldback pairing of regions that have failed to pair homologously.by D. Schweizer     

In vivo analysis of synaptonemal complex formation during yeast meiosis

During meiotic prophase a synaptonemal complex (SC) forms between each pair of homologous chromosomes and is believed to be involved in regulating recombination. Studies on SCs usually destroy nuclear architecture, making it impossible to examine the relationship of these structures to the rest of the nucleus. In Saccharomyces cerevisiae the meiosis-specific Zip1 protein is found throughout the entire length of each SC. To analyze the formation and structure of SCs in living cells, a functional ZIP1::GFP fusion was constructed and introduced into yeast. The ZIP1::GFP fusion produced fluorescent SCs and rescued the spore lethality phenotype of zip1 mutants. Optical sectioning and fluorescence deconvolution light microscopy revealed that, at zygotene, SC assembly was initiated at foci that appeared uniformly distributed throughout the nuclear volume. At early pachytene, the full-length SCs were more likely to be localized to the nuclear periphery while at later stages the SCs appeared to redistribute throughout the nuclear volume. These results suggest that SCs undergo dramatic rearrangements during meiotic prophase and that pachytene can be divided into two morphologically distinct substages: pachytene A, when SCs are perinuclear, and pachytene B, when SCs are uniformly distributed throughout the nucleus. ZIP1::GFP also facilitated the enrichment of fluorescent SC and the identification of meiosis-specific proteins by MALDI-TOF mass spectroscopy.     

Ultrastructure of the synaptic autosomes and the ZW bivalent in chicken oocytes

Chromosomal axes of chicken oocytes from pre- and post-hatching chickens were analyzed with a microspreading technique for electron microscopy. At leptotene, chromosomal axes begin to be formed as discontinuous, non-polarized axial segments. During zygotene synaptonemal complex (SC) formation begins at the axial ends attached to the nuclear envelope. Polarization of axial ends is nearly simultaneous with the beginning of SC formation. The complete SC set is found at pachytene and it consists of 38 SC's and an unequal SC which has been identified as the ZW pair. This unequal SC is formed by two axes of different length. The Z and W axes represent 6.2% and 4.5% respectively of the combined length of the SC set plus the Z axis. The unpaired segment of the Z axis shortens markedly from early to mid-pachytene and becomes thicker than the lateral elements of SCs. In the paired region the Z axis forms most of the twists around a straighter W axis, suggesting some extent of non-homologous pairing between the Z and W chromosomes in this region. The existence of partial synapsis of the Z and W axes without heteropycnosis of the sex chromosomes is in marked contrast to partial synapsis in the heteropycnotic XY body of mammalian spermatocytes.     

The synaptic behaviour of the wild forms of Triticum turgidum and T. timopheevii.

Different wild allopolyploid species of Triticeae show extensive bivalent formation at zygotene while a considerable number of multivalents is present in cultivated polyploid wheats. To study the chromosome behaviour at early meiotic stages in wild forms of tetraploid wheats Triticum turgidum and T timopheevii (2n = 4x = 28) we have analysed the synaptic pattern in fully traced spread nuclei at mid- and late zygotene and at pachytene of wild accessions of these species. The mean number of synaptonemal complex (SC) bivalents at mid-zygotene ranged from 12.22 to 13.14 among the accessions studied indicating a strong restriction of synapsis initiation to homologous chromosomes. The mean of bivalents increased at pachytene because of the transformation of multivalents into bivalents. Ring bivalents observed at metaphase I support that SC bivalents were formed by homologous chromosomes. The average values of SC bivalents at mid-zygotene in the wild forms are much higher than the average values observed in the cultivated tetraploid wheats but similar to that of a mutant line of T turgidum with a duplication that includes Ph1, the major homoeologous pairing suppressor locus. These results suggest that the efficiency of the mechanism operating in the homologous recognition for synapsis is higher in wild wheat populations than in cultivated varieties. Apparently, a relatively detrimental modification of the pairing regulating genetic system accompanied the domestication of the wild wheat forms.     

A novel mammalian HORMA domain-containing protein, HORMAD1, preferentially associates with unsynapsed meiotic chromosomes

HORMA domain-containing proteins regulate interactions between homologous chromosomes (homologs) during meiosis in a wide range of eukaryotes. We have identified a mouse HORMA domain-containing protein, HORMAD1, and biochemically and cytologically shown it to be associated with the meiotic chromosome axis. HORMAD1 first accumulates on the chromosomes during the leptotene to zygotene stages of meiotic prophase I. As germ cells progress into the pachytene stage, HORMAD1 disappears from the synapsed chromosomal regions. However, once the chromosomes desynapse during the diplotene stage, HORMAD1 again accumulates on the chromosome axis of the desynapsed homologs. HORMAD1 thus preferentially localizes to unsynapsed or desynapsed chromosomal regions during the prophase I stage of meiosis. Analysis of mutant strains lacking different components of the synaptonemal complex (SC) revealed that establishment of the SC is required for the displacement of HORMAD1 from the chromosome axis. Our results therefore strongly suggest that also mammalian cells use a HORMA domain-containing protein as part of a surveillance system that monitors synapsis or other interactions between homologs.     

The CSN/COP9 Signalosome Regulates Synaptonemal Complex Assembly during Meiotic Prophase I of Caenorhabditis elegans

The synaptonemal complex (SC) is a conserved protein structure that holds homologous chromosome pairs together throughout much of meiotic prophase I. It is essential for the formation of crossovers, which are required for the proper segregation of chromosomes into gametes. The assembly of the SC is likely to be regulated by post-translational modifications. The CSN/COP9 signalosome has been shown to act in many pathways, mainly via the ubiquitin degradation/proteasome pathway. Here we examine the role of the CSN/COP9 signalosome in SC assembly in the model organism C. elegans. Our work shows that mutants in three subunits of the CSN/COP9 signalosome fail to properly assemble the SC. In these mutants, SC proteins aggregate, leading to a decrease in proper pairing between homologous chromosomes. The reduction in homolog pairing also results in an accumulation of recombination intermediates and defects in repair of meiotic DSBs to form the designated crossovers. The effect of the CSN/COP9 signalosome mutants on synapsis and crossover formation is due to increased neddylation, as reducing neddylation in these mutants can partially suppress their phenotypes. We also find a marked increase in apoptosis in csn mutants that specifically eliminates nuclei with aggregated SC proteins. csn mutants exhibit defects in germline proliferation, and an almost complete pachytene arrest due to an inability to activate the MAPK pathway. The work described here supports a previously unknown role for the CSN/COP9 signalosome in chromosome behavior during meiotic prophase I.     

Heat stress interferes with formation of double-strand breaks and homolog synapsis

Meiotic recombination (MR) drives novel combinations of alleles and contributes to genomic diversity in eukaryotes. In this study, we showed that heat stress (36°C–38°C) over the fertile threshold fully abolished crossover formation in Arabidopsis (Arabidopsis thaliana). Cytological and genetic studies in wild-type plants and syn1 and rad51 mutants suggested that heat stress reduces generation of SPO11-dependent double-strand breaks (DSBs). In support, the abundance of recombinase DMC1, which is required for MR-specific DSB repair, was significantly reduced under heat stress. In addition, high temperatures induced disassembly and/or instability of the ASY4- but not the SYN1-mediated chromosome axis. At the same time, the ASY1-associated lateral element of the synaptonemal complex (SC) was partially affected, while the ZYP1-dependent central element of SC was disrupted, indicating that heat stress impairs SC formation. Moreover, expression of genes involved in DSB formation; e.g. SPO11-1, PRD1, 2, and 3 was not impacted; however, recombinase RAD51 and chromosome axis factors ASY3 and ASY4 were significantly downregulated under heat stress. Taken together, these findings revealed that heat stress inhibits MR via compromised DSB formation and homolog synapsis, which are possible downstream effects of the impacted chromosome axis. Our study thus provides evidence shedding light on how increasing environmental temperature influences MR in Arabidopsis.

Heat stress inhibits CO formation by affecting SPO11-dependent DSB formation and synapsis of homologous chromosomes, probably through its impact on chromosome axis.