[show abstract][hide abstract] ABSTRACT: Whole genome duplication (WGD) is a major factor in the evolution of multicellular eukaryotes, yet by doubling the number of homologs, WGD severely challenges reliable chromosome segregation [1-3], a process conserved across kingdoms . Despite this, numerous genome-duplicated (polyploid) species persist in nature, indicating early problems can be overcome [1, 2]. Little is known about which genes are involved-only one has been molecularly characterized . To gain new insights into the molecular basis of adaptation to polyploidy, we investigated genome-wide patterns of differentiation between natural diploids and tetraploids of Arabidopsis arenosa, an outcrossing relative of A. thaliana [6, 7]. We first show that diploids are not preadapted to polyploid meiosis. We then use a genome scanning approach to show that although polymorphism is extensively shared across ploidy levels, there is strong ploidy-specific differentiation in 39 regions spanning 44 genes. These are discrete, mostly single-gene peaks of sharply elevated differentiation. Among these peaks are eight meiosis genes whose encoded proteins coordinate a specific subset of early meiotic functions, suggesting these genes comprise a polygenic solution to WGD-associated chromosome segregation challenges. Our findings indicate that even conserved meiotic processes can be capable of nimble evolutionary shifts when required.
Current biology: CB 10/2013; · 10.99 Impact Factor
[show abstract][hide abstract] ABSTRACT: PRDM9 directs human meiotic crossover hot spots to intergenic sequence motifs, whereas budding yeast hot spots overlap regions of low nucleosome density (LND) in gene promoters. To investigate hot spots in plants, which lack PRDM9, we used coalescent analysis of genetic variation in Arabidopsis thaliana. Crossovers increased toward gene promoters and terminators, and hot spots were associated with active chromatin modifications, including H2A.Z, histone H3 Lys4 trimethylation (H3K4me3), LND and low DNA methylation. Hot spot-enriched A-rich and CTT-repeat DNA motifs occurred upstream and downstream, respectively, of transcriptional start sites. Crossovers were asymmetric around promoters and were most frequent over CTT-repeat motifs and H2A.Z nucleosomes. Pollen typing, segregation and cytogenetic analysis showed decreased numbers of crossovers in the arp6 H2A.Z deposition mutant at multiple scales. During meiosis, H2A.Z forms overlapping chromosomal foci with the DMC1 and RAD51 recombinases. As arp6 reduced the number of DMC1 or RAD51 foci, H2A.Z may promote the formation or processing of meiotic DNA double-strand breaks. We propose that gene chromatin ancestrally designates hot spots within eukaryotes and PRDM9 is a derived state within vertebrates.
[show abstract][hide abstract] ABSTRACT: Shugoshin is a protein conserved in eukaryotes and protects sister chromatid cohesion at centromeres in meiosis. In our study, we identified the homologs of SGO1 and SGO2 in Arabidopsis thaliana. We show that AtSGO1 is necessary for the maintenance of centromere cohesion in meiosis I since atsgo1 mutants display premature separation of sister chromatids starting from anaphase I. Furthermore, we show that the localization of the specific centromeric cohesin AtSYN1 is not affected in atsgo1, suggesting that SGO1 centromere cohesion maintenance is not mediated by protection of SYN1 from cleavage. Finally, we show that AtSGO2 is dispensable for both meiotic and mitotic cell progression in Arabidopsis.
[show abstract][hide abstract] ABSTRACT: The application of proteomics techniques to the study of plant meiosis has the potential to make a valuable contribution to our understanding of the molecular events underpinning meiotic processes. Here we describe the preparation of meiotic protein complexes from Arabidopsis thaliana and its close crop relative, Brassica oleracea, by co-immunoprecipitation for in-solution analysis by tandem mass spectrometry (MS/MS). Early results using these techniques have proved encouraging, enabling the identification of candidate AtASY1-interacting proteins in A. thaliana and providing evidence of an in planta interaction between BoASY1 and BoASY3 in B. oleracea. The detection of phospho-modified peptides of BoASY1 and BoASY3 suggests that this approach may be useful for studying meiotic protein modification events.
Methods in molecular biology (Clifton, N.J.) 01/2013; 990:215-26.
[show abstract][hide abstract] ABSTRACT: In this study we have analysed AtASY3, a coiled-coil domain protein that is required for normal meiosis in Arabidopsis. Analysis of an Atasy3-1 mutant reveals that loss of the protein compromises chromosome axis formation and results in reduced numbers of meiotic crossovers (COs). Although the frequency of DNA double-strand breaks (DSBs) appears moderately reduced in Atasy3-1, the main recombination defect is a reduction in the formation of COs. Immunolocalization studies in wild-type meiocytes indicate that the HORMA protein AtASY1, which is related to Hop1 in budding yeast, forms hyper-abundant domains along the chromosomes that are spatially associated with DSBs and early recombination pathway proteins. Loss of AtASY3 disrupts the axial organization of AtASY1. Furthermore we show that the AtASY3 and AtASY1 homologs BoASY3 and BoASY1, from the closely related species Brassica oleracea, are co-immunoprecipitated from meiocyte extracts and that AtASY3 interacts with AtASY1 via residues in its predicted coiled-coil domain. Together our results suggest that AtASY3 is a functional homolog of Red1. Since studies in budding yeast indicate that Red1 and Hop1 play a key role in establishing a bias to favor inter-homolog recombination (IHR), we propose that AtASY3 and AtASY1 may have a similar role in Arabidopsis. Loss of AtASY3 also disrupts synaptonemal complex (SC) formation. In Atasy3-1 the transverse filament protein AtZYP1 forms small patches rather than a continuous SC. The few AtMLH1 foci that remain in Atasy3-1 are found in association with the AtZYP1 patches. This is sufficient to prevent the ectopic recombination observed in the absence of AtZYP1, thus emphasizing that in addition to its structural role the protein is important for CO formation.
[show abstract][hide abstract] ABSTRACT: Following successful completion of the Brassica rapa sequencing project, the next step is to investigate functions of individual genes/proteins. For Arabidopsis thaliana, large amounts of protein-protein interaction (PPI) data are available from the major PPI databases (DBs). It is known that Brassica crop species are closely related to A. thaliana. This provides an opportunity to infer the B. rapa interactome using PPI data available from A. thaliana. In this paper, we present an inferred B. rapa interactome that is based on the A. thaliana PPI data from two resources: (i) A. thaliana PPI data from three major DBs, BioGRID, IntAct, and TAIR. (ii) ortholog-based A. thaliana PPI predictions. Linking between B. rapa and A. thaliana was accomplished in three complementary ways: (i) ortholog predictions, (ii) identification of gene duplication based on synteny and collinearity, and (iii) BLAST sequence similarity search. A complementary approach was also applied, which used known/predicted domain-domain interaction data. Specifically, since the two species are closely related, we used PPI data from A. thaliana to predict interacting domains that might be conserved between the two species. The predicted interactome was investigated for the component that contains known A. thaliana meiotic proteins to demonstrate its usability.
[show abstract][hide abstract] ABSTRACT: Meiosis is a central feature of sexual reproduction. Studies in plants have made and continue to make an important contribution to fundamental research aimed at the understanding of this complex process. Moreover, homologous recombination during meiosis provides the basis for plant breeders to create new varieties of crops. The increasing global demand for food, combined with the challenges from climate change, will require sustained efforts in crop improvement. An understanding of the factors that control meiotic recombination has the potential to make an important contribution to this challenge by providing the breeder with the means to make fuller use of the genetic variability that is available within crop species. Cytogenetic studies in plants have provided considerable insights into chromosome organization and behaviour during meiosis. More recently, studies, predominantly in Arabidopsis thaliana, are providing important insights into the genes and proteins that are required for crossover formation during plant meiosis. As a result, substantial progress in the understanding of the molecular mechanisms that underpin meiosis in plants has begun to emerge. This article summarizes current progress in the understanding of meiotic recombination and its control in Arabidopsis. We also assess the relationship between meiotic recombination in Arabidopsis and other eukaryotes, highlighting areas of close similarity and apparent differences.
New Phytologist 03/2011; 190(3):523-44. · 6.74 Impact Factor
[show abstract][hide abstract] ABSTRACT: We have analysed the role of RBR (retinoblastoma related), the Arabidopsis homologue of the tumour suppressor Retinoblastoma protein (pRb), during meiosis. We characterise the rbr-2 mutation, which causes a loss of RBR in male meiocytes. The rbr-2 plants exhibit strongly reduced fertility, while vegetative growth is generally unaffected. The reduced fertility is due to a meiotic defect that results in reduced chiasma formation and subsequent errors in chromosome disjunction. Immunolocalisation studies in wild-type meiocytes reveal that RBR is recruited as foci to the chromosomes during early prophase I in a DNA double-strand-break-dependent manner. In the absence of RBR, expression of several meiotic genes is reduced. The localisation of the recombinases AtRAD51 and AtDMC1 is normal. However, localisation of the MutS homologue AtMSH4 is compromised. Additionally, polymerisation of the synaptonemal complex protein AtZYP1 is abnormal. Together, these data indicate that loss of RBR during meiosis results in a reduction of crossover formation and an associated failure in chromosome synapsis. Our results indicate that RBR has an important role in meiosis affecting different aspects of this complex process.
The EMBO Journal 02/2011; 30(4):744-55. · 9.82 Impact Factor
[show abstract][hide abstract] ABSTRACT: Replication protein A (RPA) is involved in many aspects of DNA metabolism including meiotic recombination. Many species possess a single RPA1 gene but Arabidopsis possesses five RPA1 paralogues. This feature has enabled us to gain further insight into the meiotic role of RPA1. Proteomic analysis implicated one of the AtRPA1 family (AtRPA1a) in meiosis. Immunofluorescence studies confirmed that AtRPA1a is associated with meiotic chromosomes from leptotene through to early pachytene. Analysis of an Atrpa1a mutant revealed that AtRPA1a is not essential at early stages in the recombination pathway. DNA double-strand breaks are repaired in Atrpa1a, but the mutant is defective in the formation of crossovers, exhibiting a 60% reduction in chiasma frequency. Consistent with this, localization of recombination proteins AtRAD51 and AtMSH4 appears normal, whereas the numbers of AtMLH1 and AtMLH3 foci at pachytene are significantly reduced. This suggests that the defect in Atrpa1a is manifested at the stage of second-end capture. Analysis of Atrpa1a/Atmsh4 and Atrpa1a/Atmlh3 double mutants indicates that loss of AtRPA1a predominantly affects the formation of class I, interference-dependent crossovers.
The EMBO Journal 02/2009; 28(4):394-404. · 9.82 Impact Factor
[show abstract][hide abstract] ABSTRACT: Advances in molecular biology and in the genetics of Arabidopsis thaliana have led to this organism becoming an important model for the analysis of meiosis in plants. Cytogenetic investigations are pivotal to meiotic studies and a number of technological improvements for Arabidopsis cytology have provided a range of tools to investigate chromosome behaviour during meiosis. This chapter includes protocols on basic cytology, FISH analysis, immunocytology, a procedure for a meiotic time course and electron microscopy.
Methods in molecular biology (Clifton, N.J.) 02/2009; 558:131-45.
[show abstract][hide abstract] ABSTRACT: MSH5, a meiosis-specific member of the MutS-homologue family of genes, is required for normal levels of recombination in budding yeast, mouse and Caenorhabditis elegans. In this paper we report the identification and characterization of the Arabidopsis homologue of MSH5 (AtMSH5). Transcripts of AtMSH5 are specific to reproductive tissues, and immunofluorescence studies indicate that expression of the protein is abundant during prophase I of meiosis. In a T-DNA tagged insertional mutant (Atmsh5-1), recombination is reduced to about 13% of wild-type levels. The residual chiasmata are randomly distributed between cells and chromosomes. These data provide further evidence for at least two pathways of meiotic recombination in Arabidopsis and indicate that AtMSH5 protein is required for the formation of class I interference-sensitive crossovers. Localization of AtMSH5 to meiotic chromosomes occurs at leptotene and is dependent on DNA double-strand break formation and strand exchange. Localization of AtMSH5 to the chromatin at mid-prophase I is dependent on expression of AtMSH4. At late zygotene/early pachytene a proportion of AtMSH5 foci co-localize with AtMLH1 which marks crossover-designated sites. Chromosome synapsis appears to proceed normally, without significant delay, in Atmsh5-1 but the pachytene stage is extended by several hours, indicative of the operation of a surveillance system that monitors the progression of prophase I.
The Plant Journal 08/2008; 55(1):28-39. · 6.58 Impact Factor
[show abstract][hide abstract] ABSTRACT: Meiotic crossovers/chiasmata, that are required to ensure chromosome disjunction, arise via the class I interference-dependent pathway or via the class II interference-free pathway. The proportions of these two classes vary considerably between different organisms. In Arabidopsis, about 85% of chiasmata are eliminated in Atmsh4 mutants, denoting that these are class I events. In budding and fission yeasts Msh4-independent crossovers arise largely or entirely via a Mus81-dependent pathway. To investigate the origins of the 15% residual (AtMSH4-independent) chiasmata in Arabidopsis we conducted a cytological and molecular analysis of AtMUS81 meiotic expression and function. Although AtMUS81 functions in somatic DNA repair and recombination, it is more highly expressed in reproductive tissues. The protein is abundantly present in early prophase I meiocytes, where it co-localizes, in a double-strand break-dependent manner, with the recombination protein AtRAD51. Despite this, an Atmus81 mutant shows normal growth and has no obvious defects in reproductive development that would indicate meiotic impairment. A cytological analysis confirmed that meiosis was apparently normal in this mutant and its mean chiasma frequency was similar to that of wild-type plants. However, an Atmsh4/Atmus81 double mutant revealed a significantly reduced mean chiasma frequency (0.85 per cell), compared with an Atmsh4 single mutant (1.25 per cell), from which we conclude that AtMUS81 accounts for some, but not all, of the 15% AtMSH4-independent residual crossovers. It is possible that other genes are responsible for these residual chiasmata. Alternatively the AtMUS81 pathway coexists with an alternative parallel pathway that can perform the same functions.
The Plant Journal 05/2008; 54(1):152-62. · 6.58 Impact Factor
[show abstract][hide abstract] ABSTRACT: Meiosis is a fundamental and evolutionarily conserved process that is central to the life cycles of all sexually reproducing eukaryotes. An understanding of this process is critical to furthering research on reproduction, fertility, genetics and breeding. Plants have been used extensively in cytogenetic studies of meiosis during the last century. Until recently, our knowledge of the molecular and functional aspects of meiosis has emerged from the study of non-plant model organisms, especially budding yeast. However, the emergence of Arabidopsis thaliana as the model organism for plant molecular biology and genetics has enabled significant progress in the characterisation of key genes and proteins controlling plant meiosis. The development of molecular and cytological techniques in Arabidopsis, besides allowing investigation of the more conserved aspects of meiosis, are also providing insights into features of this complex process which may vary between organisms. This review highlights an example of this recent progress by focussing on ASY1, a meiosis-specific Arabidopsis protein which shares some similarity with the N-terminus region of the yeast axial core-associated protein, HOP1, a component of a multiprotein complex which acts as a meiosis-specific barrier to sister-chromatid repair in budding yeast. In the absence of ASY1, synapsis is interrupted and chiasma formation is dramatically reduced. ASY1 protein is initially detected during early meiotic G2 as numerous foci distributed over the chromatin. As G2 progresses the signal appears to be increasingly continuous and is closely associated with the axial elements. State-of-the-art cytogenetic techniques have revealed that initiation of recombination is synchronised with the formation of the chromosome axis. Furthermore, in the context of the developing chromosome axes, ASY1 plays a crucial role in co-ordinating the activity of a key member of the homologous recombination machinery, AtDMC1.
Cytogenetic and Genome Research 02/2008; 120(3-4):302-12. · 1.84 Impact Factor
[show abstract][hide abstract] ABSTRACT: Arabidopsis has emerged as an excellent system for meiosis research due to the development of powerful
cytological, genetical and molecular methodologies combined with particular biological features of plant
meiosis. This chapter reviews these developments and highlights their impact on the investigation of meiotic
recombination. We focus specifically on meiotic recombination, but it is important to bear in mind that
research on somatic DNA repair and recombination in Arabidopsis is ahighly active field. Indeed,
many aspects of somatic and meiotic recombination overlap and several genes are involved in both processes.
An overview of the features of meiotic recombination in Arabidopsis is obtainable from cytogenetic
analysis of chiasma frequency and distribution and also from high-throughput genetic linkage analysis using
molecular markers. Recombination parameters derived from these different approaches are in excellent agreement.
Biotic and abiotic factors have been shown to influence recombination frequency in Arabidopsis. Forward
and reverse genetic procedures have played prominent roles in the functional analysis of meiosis in Arabidopsis.
Alarge and growing number of genes that are directly or indirectly involved in the meiotic recombination
process and its regulation have been identified and analysed. These range from genes involved in the initiation
of recombination through programmed double strand breaks, through the processing of these events as recombination
intermediates to their final emergence as mature reciprocal crossover events (COs). Although the molecular
events of recombination are largely conserved across eukaryotes, comparisons with other model species indicate
some interesting though poorly understood differences. In common with other systems that have been analysed
it is clear that the regulation of meiotic recombination in Arabidopsis involves acarefully programmed
progressive selection of arelatively small number of recombination initiations to become COs. Evidence
is also presented for the existence of more than one pathway to COs in Arabidopsis as in some other eukaryote
systems. Finally, meiosis is increasingly being viewed as ahighly complex series of interrelated
events that are necessarily closely coordinated. In this context there is an emerging understanding, from
Arabidopsis and other species, that recombination proceeds in the context of complex alterations in chromatin
organization and that meiotic progression is dependent on the correct sequence and timing of these events.
[show abstract][hide abstract] ABSTRACT: Immunocytochemistry reveals that the Arabidopsis mismatch repair proteins AtMSH4, AtMLH3 and AtMLH1 are expressed during prophase I of meiosis. Expression of AtMSH4 precedes AtMLH3 and AtMLH1 which co-localize as foci during pachytene. Co-localization between AtMSH4 and AtMLH3 occurs, but appears transient. AtMLH3 foci are not detected in an Atmsh4 mutant. However, localization of AtMSH4 is unaffected in Atmlh3, suggesting that recombination may proceed to dHj (double Holliday junction) formation. Mean chiasma frequency in Atmsh4 is reduced to 1.55 compared with 9.86 in wild-type. In contrast with wild-type, the distribution of residual crossovers in Atmsh4 closely fits a Poisson distribution. This is consistent with a two-pathway model for meiotic crossing-over whereby most crossovers occur via an AtMSH4-dependent pathway that is subject to interference, with the remaining crossovers arising via an interference-independent pathway. Loss of AtMLH3 results in an approx. 60% reduction in crossovers. Results suggest that dHj resolution can occur, but in contrast with wild-type where most or all dHjs are directed to form crossovers, the outcome is biased in favour of a non-crossover outcome. The results are compatible with a model whereby the MutL complex maintains or imposes a dHj conformation that ensures crossover formation.
Biochemical Society Transactions 09/2006; 34(Pt 4):542-4. · 2.59 Impact Factor
[show abstract][hide abstract] ABSTRACT: During meiosis, crossing-over--the exchange of genetic material between maternal and paternal chromosomes--is stringently controlled to restrict the number of crossovers per chromosome pair. In this issue of Cell, Martini et al., (2006) report that the reduction of crossover-initiating events does not result in fewer crossovers. These results have important implications for our understanding of crossover control.
[show abstract][hide abstract] ABSTRACT: With respect to history, plants have provided an ideal system for cytogenetical analysis of the synaptonemal complex (SC). However, until recently, the identification of the genes that encode the SC in plants has proved elusive. In recent years, Arabidopsis thaliana was developed as a model system for plant meiosis research. As a result, there was substantial progress in the isolation of meiotic genes and this has recently led to the isolation of the first plant SC gene, ZYP1. The ZYP1 gene encodes a transverse filament (TF) protein that is predicted to have structural similarity to TF proteins found in other organisms. Analysis of plants deficient in ZYP1 expression has provided important insights into the function of the SC in plants. Loss of ZYP1 has only a limited effect on the overall level of recombination. However, it is associated with extensive nonhomologous recombination leading to multivalent formation at metaphase I. This phenomenon was not previously reported in other organisms. It is important to note that cytological analysis of the ZYP1 deficient lines indicates that SC formation is not required for the imposition of crossover interference.
[show abstract][hide abstract] ABSTRACT: Ribonuclease assays have revealed, in contrast to the self-incompatibility (SI) system of Nicotiana alata, there is no detectable ribonuclease activity that correlates with the presence of the functional stigmatic S-gene product in Papaver rhoeas. Thus, we have shown that the inhibition of incompatible pollen tube growth in P. rhoeas is not associated with ribonuclease activity. Furthermore, the finding that pollen from P. rhoeas, unlike that from N. alata, is insensitive to purified bovine pancreatic ribonuclease A at very high concentrations, suggests that the involvement of ribonucleases in the inhibition reaction of the SI response in P. rhoeas is unlikely. In addition, the level of ribonuclease activity in mature stigmas of P. rhoeas is very much lower than that in N. alata and significantly, the level of ribonuclease activity did not rise in conjunction with the developmental expression of SI. Therefore, as a result of these studies, we believe that SI in P. rhoeas does not involve ribonuclease activity.
[show abstract][hide abstract] ABSTRACT: The duplicated Arabidopsis genes ZYP1a/ZYP1b encode closely related proteins with structural similarity to the synaptonemal complex (SC) transverse filament proteins from other species. Immunolocalization detects ZYP1 foci at late leptotene, which lengthen until at pachytene fluorescent signals extending the entire length of the fully synapsed homologs are observed. Analysis of zyp1a and zyp1b T-DNA insertion mutants indicates that the proteins are functionally redundant. The SC is not formed in the absence of ZYP1 and prophase I progression is significantly delayed suggesting the existence of an intraprophase I surveillance mechanism. Recombination is only slightly reduced in the absence of ZYP1 such that the chiasma frequency at metaphase I is approximately 80% of wild type. Moreover cytological analysis indicates that chiasma distribution within zyp1 bivalents is indistinguishable from wild type, providing evidence that the SC is not required for the imposition of interference. Importantly in the absence of ZYP1, recombination occurs between both homologous and nonhomologous chromosomes suggesting the protein is required to ensure the fidelity of meiotic chromosome associations.
Genes & Development 11/2005; 19(20):2488-500. · 12.44 Impact Factor
[show abstract][hide abstract] ABSTRACT: The analysis of meiosis in higher plants has benefited considerably in recent years from the completion of the genome sequence of the model plant Arabidopsis thaliana and the development of cytological techniques for this species. A combination of forward and reverse genetics has provided important routes toward the identification of meiotic genes in Arabidopsis. Nevertheless identification of certain meiotic genes remains a challenge due to problems such as limited sequence conservation between species, existence of closely related gene families and in some cases functional redundancy between gene family members. Hence there is a requirement to develop new experimental approaches that can be used in conjunction with existing methods to enable a greater range of plant meiotic genes to be identified. As one potential route towards this goal we have initiated a proteomics-based approach. Unfortunately, the small size of Arabidopsis anthers makes an analysis in this species technically very difficult. Therefore we have initially focussed on Brassica oleracea which is closely related to Arabidopsis, but has the advantage of possessing significantly larger anthers. The basic strategy has been to use peptide mass-finger printing and matrix-assisted laser desorption ionization time of flight mass spectrometry to analyse proteins expressed in meiocytes during prophase I of meiosis. Initial experiments based on the analysis of proteins from staged anther tissue proved disappointing due to the low level of detection of proteins associated with meiosis. However, by extruding meiocytes in early prophase I from individual anthers prior to analysis a significant enrichment of meiotic proteins has been achieved. Analysis suggests that at least 18% of the proteins identified by this route have a putative meiotic function and that this figure could be as high as one-third of the total. Approaches to increase the enrichment of proteins involved in meiotic recombination and chromosome synapsis are also described.
Cytogenetic and Genome Research 02/2005; 109(1-3):181-9. · 1.84 Impact Factor