Heat stress impairs centromere structure and segregation of meiotic chromosomes in Arabidopsis.

Heat stress is a major threat to global crop production, and understanding its impact on plant fertility is crucial for developing climate-resilient crops. Despite the known negative effects of heat stress on plant reproduction, the underlying molecular mechanisms remain poorly understood. Here, we investigated the impact of elevated temperature on centromere structure and chromosome segregation during meiosis in Arabidopsis thaliana. Consistent with previous studies, heat stress leads to a decline in fertility and micronuclei formation in pollen mother cells. Our results reveal that elevated temperature causes a decrease in the amount of centromeric histone and the kinetochore protein BMF1 at meiotic centromeres with increasing temperature. Furthermore, we show that heat stress increases the duration of meiotic divisions and prolongs the activity of the spindle assembly checkpoint during meiosis I, indicating an impaired efficiency of the kinetochore attachments to spindle microtubules. Our analysis of mutants with reduced levels of centromeric histone suggests that weakened centromeres sensitize plants to elevated temperature, resulting in meiotic defects and reduced fertility even at moderate temperatures. These results indicate that the structure and functionality of meiotic centromeres in Arabidopsis are highly sensitive to heat stress, and suggest that centromeres and kinetochores may represent a critical bottleneck in plant adaptation to increasing temperatures.

Barley stripe mosaic virus-mediated somatic and heritable gene editing in barley (Hordeum vulgare L.).

Genome editing strategies in barley (Hordeum vulgare L.) typically rely on Agrobacterium-mediated genetic transformation for the delivery of required genetic reagents involving tissue culture techniques. These approaches are genotype-dependent, time-consuming, and labor-intensive, which hampers rapid genome editing in barley. More recently, plant RNA viruses have been engineered to transiently express short guide RNAs facilitating CRISPR/Cas9-based targeted genome editing in plants that constitutively express Cas9. Here, we explored virus-induced genome editing (VIGE) based on barley stripe mosaic virus (BSMV) in Cas9-transgenic barley. Somatic and heritable editing in the ALBOSTRIANS gene (CMF7) resulting in albino/variegated chloroplast-defective barley mutants is shown. In addition, somatic editing in meiosis-related candidate genes in barley encoding ASY1 (an axis-localized HORMA domain protein), MUS81 (a DNA structure-selective endonuclease), and ZYP1 (a transverse filament protein of the synaptonemal complex) was achieved. Hence, the presented VIGE approach using BSMV enables rapid somatic and also heritable targeted gene editing in barley.

TurboID-based proteomic profiling of meiotic chromosome axes in Arabidopsis thaliana.

During meiotic prophase I, sister chromatids are arranged in a loop-base array along a proteinaceous structure, called the meiotic chromosome axis. This structure is essential for synapsis and meiotic recombination progression and hence formation of genetically diverse gametes. Proteomic studies in plants aiming to unravel the composition and regulation of meiotic axes are constrained by limited meiotic cells embedded in floral organs. Here we report TurboID (TbID)-based proximity labelling (PL) in meiotic cells of Arabidopsis thaliana. TbID fusion to the two meiotic chromosome axis proteins ASY1 and ASY3 enabled the identification of their proximate 'interactomes' based on affinity purification coupled with mass spectrometry. We identified 39 ASY1 and/or ASY3 proximate candidates covering most known chromosome axis-related proteins. Functional studies of selected candidates demonstrate that not only known meiotic candidates but also new meiotic proteins were uncovered. Hence, TbID-based PL in meiotic cells enables the identification of chromosome axis proximate proteins in A. thaliana.

High temperature increases centromere-mediated genome elimination frequency and enhances haploid induction in Arabidopsis.

Double haploid production is the most effective way to create true-breeding lines in a single generation. In Arabidopsis, haploid induction via mutation of the centromere-specific histone H3 (cenH3) has been shown when the mutant is outcrossed to the wild-type, and the wild-type genome remains in the haploid progeny. However, factors that affect haploid induction are still poorly understood. Here, we report that a mutant of the cenH3 assembly factor Kinetochore Null2 (KNL2) can be used as a haploid inducer when pollinated by the wild-type. We discovered that short-term temperature stress of the knl2 mutant increased the efficiency of haploid induction 10-fold. We also demonstrated that a point mutation in the CENPC-k motif of KNL2 is sufficient to generate haploid-inducing lines, suggesting that haploid-inducing lines in crops can be identified in a naturally occurring or chemically induced mutant population, avoiding the generic modification (GM) approach at any stage. Furthermore, a cenh3-4 mutant functioned as a haploid inducer in response to short-term heat stress, even though it did not induce haploids under standard conditions. Thus, we identified KNL2 as a new target gene for the generation of haploid-inducer lines and showed that exposure of centromeric protein mutants to high temperature strongly increases their haploid induction efficiency.

The meiotic topoisomerase VI B subunit (MTOPVIB) is essential for meiotic DNA double-strand break formation in barley (Hordeum vulgare L.).

KEY MESSAGE: In barley (Hordeum vulgare), MTOPVIB is critical for meiotic DSB and accompanied SC and CO formation while dispensable for meiotic bipolar spindle formation. Homologous recombination during meiosis assures genetic variation in offspring. Programmed meiotic DNA double-strand breaks (DSBs) are repaired as crossover (CO) or non-crossover (NCO) during meiotic recombination. The meiotic topoisomerase VI (TopoVI) B subunit (MTOPVIB) plays an essential role in meiotic DSB formation critical for CO-recombination. More recently MTOPVIB has been also shown to play a role in meiotic bipolar spindle formation in rice and maize. Here, we describe a meiotic DSB-defective mutant in barley (Hordeum vulgare L.). CRISPR-associated 9 (Cas9) endonuclease-generated mtopVIB plants show complete sterility due to the absence of meiotic DSB, synaptonemal complex (SC), and CO formation leading to the occurrence of univalents and their unbalanced segregation into aneuploid gametes. In HvmtopVIB plants, we also frequently found the bi-orientation of sister kinetochores in univalents during metaphase I and the precocious separation of sister chromatids during anaphase I. Moreover, the near absence of polyads after meiosis II, suggests that despite being critical for meiotic DSB formation in barley, MTOPVIB seems not to be strictly required for meiotic bipolar spindle formation.

Image analysis workflows to reveal the spatial organization of cell nuclei and chromosomes.

Nucleus, chromatin, and chromosome organization studies heavily rely on fluorescence microscopy imaging to elucidate the distribution and abundance of structural and regulatory components. Three-dimensional (3D) image stacks are a source of quantitative data on signal intensity level and distribution and on the type and shape of distribution patterns in space. Their analysis can lead to novel insights that are otherwise missed in qualitative-only analyses. Quantitative image analysis requires specific software and workflows for image rendering, processing, segmentation, setting measurement points and reference frames and exporting target data before further numerical processing and plotting. These tasks often call for the development of customized computational scripts and require an expertise that is not broadly available to the community of experimental biologists. Yet, the increasing accessibility of high- and super-resolution imaging methods fuels the demand for user-friendly image analysis workflows. Here, we provide a compendium of strategies developed by participants of a training school from the COST action INDEPTH to analyze the spatial distribution of nuclear and chromosomal signals from 3D image stacks, acquired by diffraction-limited confocal microscopy and super-resolution microscopy methods (SIM and STED). While the examples make use of one specific commercial software package, the workflows can easily be adapted to concurrent commercial and open-source software. The aim is to encourage biologists lacking custom-script-based expertise to venture into quantitative image analysis and to better exploit the discovery potential of their images.Abbreviations: 3D FISH: three-dimensional fluorescence in situ hybridization; 3D: three-dimensional; ASY1: ASYNAPTIC 1; CC: chromocenters; CO: Crossover; DAPI: 4',6-diamidino-2-phenylindole; DMC1: DNA MEIOTIC RECOMBINASE 1; DSB: Double-Strand Break; FISH: fluorescence in situ hybridization; GFP: GREEN FLUORESCENT PROTEIN; HEI10: HUMAN ENHANCER OF INVASION 10; NCO: Non-Crossover; NE: Nuclear Envelope; Oligo-FISH: oligonucleotide fluorescence in situ hybridization; RNPII: RNA Polymerase II; SC: Synaptonemal Complex; SIM: structured illumination microscopy; ZMM (ZIP: MSH4: MSH5 and MER3 proteins); ZYP1: ZIPPER-LIKE PROTEIN 1.

Recombination landscape divergence between populations is marked by larger low-recombining regions in domesticated rye.

The genomic landscape of recombination plays an essential role in evolution. Patterns of recombination are highly variable along chromosomes, between sexes, individuals, populations, and species. In many eukaryotes, recombination rates are elevated in sub-telomeric regions and drastically reduced near centromeres, resulting in large low-recombining (LR) regions. The processes of recombination are influenced by genetic factors, such as different alleles of genes involved in meiosis and chromatin structure, as well as external environmental stimuli like temperature and overall stress. In this work, we focused on the genomic landscapes of recombination in a collection of 916 rye (Secale cereale) individuals. By analysing population structure among individuals of different domestication status and geographic origin, we detected high levels of admixture, reflecting the reproductive biology of a self-incompatible, wind-pollinating grass species. We then analysed patterns of recombination in overlapping subpopulations, which revealed substantial variation in the physical size of LR regions, with a tendency for larger LR regions in domesticated subpopulations. Genome-wide association scans (GWAS) for LR region size revealed a major quantitative-trait-locus (QTL) at which, among 18 annotated genes, an ortholog of histone H4 acetyltransferase ESA1 was located. Rye individuals belonging to domesticated subpopulations showed increased synaptonemal complex length, but no difference in crossover frequency, indicating that only the recombination landscape is different. Furthermore, the genomic region harbouring rye ScESA1 showed moderate patterns of selection in domesticated subpopulations, suggesting that larger LR regions were indirectly selected for during domestication to achieve more homogeneous populations for agricultural use.

High-throughput measuring of meiotic recombination rates in barley pollen nuclei using Crystal Digital PCRTM.

Breeding exploits novel allelic combinations assured by meiotic recombination. Barley (Hordeum vulgare) single pollen nucleus genotyping enables measurement of meiotic recombination rates in gametes before fertilization without the need for segregating populations. However, so far, established methods rely on whole-genome amplification of every single pollen nucleus due to their limited DNA content, thus restricting the number of analyzed samples. In this study, high-throughput measurements of meiotic recombination rates in barley pollen nuclei without whole-genome amplification were performed through a Crystal Digital PCRTM -based genotyping assay. Meiotic recombination rates within two centromeric and two distal chromosomal intervals were measured in hybrid plants by genotyping a total of >42 000 individual pollen nuclei (up to 4900 nuclei analyzed per plant). Determined recombination frequencies in pollen nuclei were similar to frequencies in segregating populations. We improved the efficiency of the genotyping by pretreating the pollen nuclei with a thermostable restriction enzyme. Additional opportunities for a higher sample throughput and a further increase of the genotyping efficiency are presented and discussed. Taken together, single barley pollen nucleus genotyping based on Crystal Digital PCRTM enables reliable, rapid and high-throughput meiotic recombination measurements within defined chromosomal intervals of intraspecific hybrid plants. The successful encapsulation of nuclei from a range of species with different nuclear and genome sizes suggests that the proposed method is broadly applicable to genotyping single nuclei.

Meiotic chromosome axis remodelling is critical for meiotic recombination in Brassica rapa.

Meiosis generates genetic variation through homologous recombination (HR) that is harnessed during breeding. HR occurs in the context of meiotic chromosome axes and the synaptonemal complex. To study the role of axis remodelling in crossover (CO) formation in a crop species, we characterized mutants of the axis-associated protein ASY1 and the axis-remodelling protein PCH2 in Brassica rapa. asy1 plants form meiotic chromosome axes that fail to synapse. CO formation is almost abolished, and residual chiasmata are proportionally enriched in terminal chromosome regions, particularly in the nucleolar organizing region (NOR)-carrying chromosome arm. pch2 plants show impaired ASY1 loading and remodelling, consequently achieving only partial synapsis, which leads to reduced CO formation and loss of the obligatory CO. PCH2-independent chiasmata are proportionally enriched towards distal chromosome regions. Similarly, in Arabidopsis pch2, COs are increased towards telomeric regions at the expense of (peri-) centromeric COs compared with the wild type. Taken together, in B. rapa, axis formation and remodelling are critical for meiotic fidelity including synapsis and CO formation, and in asy1 and pch2 CO distributions are altered. While asy1 plants are sterile, pch2 plants are semi-sterile and thus PCH2 could be an interesting target for breeding programmes.

Changing local recombination patterns in Arabidopsis by CRISPR/Cas mediated chromosome engineering.

Chromosomal inversions are recurrent rearrangements that occur between different plant isolates or cultivars. Such inversions may underlie reproductive isolation in evolution and represent a major obstacle for classical breeding as no crossovers can be observed between inverted sequences on homologous chromosomes. The heterochromatic knob (hk4S) on chromosome 4 is the most well-known inversion of Arabidopsis. If a knob carrying accession such as Col-0 is crossed with a knob-less accession such as Ler-1, crossovers cannot be recovered within the inverted region. Our work shows that by egg-cell specific expression of the Cas9 nuclease from Staphylococcus aureus, a targeted reversal of the 1.1 Mb long hk4S-inversion can be achieved. By crossing Col-0 harbouring the rearranged chromosome 4 with Ler-1, meiotic crossovers can be restored into a region with previously no detectable genetic exchange. The strategy of somatic chromosome engineering for breaking genetic linkage has huge potential for application in plant breeding.

MutS homologue 4 and MutS homologue 5 Maintain the Obligate Crossover in Wheat Despite Stepwise Gene Loss following Polyploidization.

Crossovers (COs) ensure accurate chromosome segregation during meiosis while creating novel allelic combinations. Here, we show that allotetraploid (AABB) durum wheat (Triticum turgidum ssp. durum) utilizes two pathways of meiotic recombination. The class I pathway requires MSH4 and MSH5 (MutSγ) to maintain the obligate CO/chiasma and accounts for ∼85% of meiotic COs, whereas the residual ∼15% are consistent with the class II CO pathway. Class I and class II chiasmata are skewed toward the chromosome ends, but class II chiasmata are significantly more distal than class I chiasmata. Chiasma distribution does not reflect the abundance of double-strand breaks, detected by proxy as RAD51 foci at leptotene, but only ∼2.3% of these sites mature into chiasmata. MutSγ maintains the obligate chiasma despite a 5.4-kb deletion in MSH5B rendering it nonfunctional, which occurred early in the evolution of tetraploid wheat and was then domesticated into hexaploid (AABBDD) common wheat (Triticum aestivum), as well as an 8-kb deletion in MSH4D in hexaploid wheat, predicted to create a nonfunctional pseudogene. Stepwise loss of MSH5B and MSH4D following hybridization and whole-genome duplication may have occurred due to gene redundancy (as functional copies of MSH5A, MSH4A, and MSH4B are still present in the tetraploid and MSH5A, MSH5D, MSH4A, and MSH4B are present in the hexaploid) or as an adaptation to modulate recombination in allopolyploid wheat.

Super-Resolution Microscopy Reveals Diversity of Plant Centromere Architecture.

Centromeres are essential for proper chromosome segregation to the daughter cells during mitosis and meiosis. Chromosomes of most eukaryotes studied so far have regional centromeres that form primary constrictions on metaphase chromosomes. These monocentric chromosomes vary from point centromeres to so-called "meta-polycentromeres", with multiple centromere domains in an extended primary constriction, as identified in Pisum and Lathyrus species. However, in various animal and plant lineages centromeres are distributed along almost the entire chromosome length. Therefore, they are called holocentromeres. In holocentric plants, centromere-specific proteins, at which spindle fibers usually attach, are arranged contiguously (line-like), in clusters along the chromosomes or in bands. Here, we summarize findings of ultrastructural investigations using immunolabeling with centromere-specific antibodies and super-resolution microscopy to demonstrate the structural diversity of plant centromeres. A classification of the different centromere types has been suggested based on the distribution of spindle attachment sites. Based on these findings we discuss the possible evolution and advantages of holocentricity, and potential strategies to segregate holocentric chromosomes correctly.

Imaging plant germline differentiation within Arabidopsis flowers by light sheet microscopy.

In higher plants, germline differentiation occurs during a relatively short period within developing flowers. Understanding of the mechanisms that govern germline differentiation lags behind other plant developmental processes. This is largely because the germline is restricted to relatively few cells buried deep within floral tissues, which makes them difficult to study. To overcome this limitation, we have developed a methodology for live imaging of the germ cell lineage within floral organs of Arabidopsis using light sheet fluorescence microscopy. We have established reporter lines, cultivation conditions, and imaging protocols for high-resolution microscopy of developing flowers continuously for up to several days. We used multiview imagining to reconstruct a three-dimensional model of a flower at subcellular resolution. We demonstrate the power of this approach by capturing male and female meiosis, asymmetric pollen division, movement of meiotic chromosomes, and unusual restitution mitosis in tapetum cells. This method will enable new avenues of research into plant sexual reproduction.

In Planta Delivery of Chemical Compounds into Barley Meiocytes: EdU as Compound Example.

Here, we describe a protocol for in planta delivery of chemical compounds into meiocytes of different barley genotypes not impacting plant fertility allowing to harvest seeds from treated plants. Compound uptake into meiocytes is assessed by determining 5-ethynyl-2'-deoxyuridine (EdU) incorporation. Similar to EdU, other compounds being soluble in an aqueous solution can be delivered in planta before/during meiosis to decipher their impact on meiosis and meiotic recombination.We give practical advice on how to deliver EdU as compound example (delivery via injection or needle and thread, addition of detergents or surfactants to increase compound uptake), how in planta compound delivery can be established for your plant material under specific growing conditions, how to generate and characterize barley hybrid plants, and how to conduct a meiotic cytological study of (treated) barley plants.

The H3 histone chaperone NASPSIM3 escorts CenH3 in Arabidopsis.

Centromeres define the chromosomal position where kinetochores form to link the chromosome to microtubules during mitosis and meiosis. Centromere identity is determined by incorporation of a specific histone H3 variant termed CenH3. As for other histones, escort and deposition of CenH3 must be ensured by histone chaperones, which handle the non-nucleosomal CenH3 pool and replenish CenH3 chromatin in dividing cells. Here, we show that the Arabidopsis orthologue of the mammalian NUCLEAR AUTOANTIGENIC SPERM PROTEIN (NASP) and Schizosaccharomyces pombe histone chaperone Sim3 is a soluble nuclear protein that binds the histone variant CenH3 and affects its abundance at the centromeres. NASPSIM3 is co-expressed with Arabidopsis CenH3 in dividing cells and binds directly to both the N-terminal tail and the histone fold domain of non-nucleosomal CenH3. Reduced NASPSIM3 expression negatively affects CenH3 deposition, identifying NASPSIM3 as a CenH3 histone chaperone.

Deregulated Phosphorylation of CENH3 at Ser65 Affects the Development of Floral Meristems in Arabidopsis thaliana.

Several histone variants are posttranslationally phosphorylated. Little is known about phosphorylation of the centromere-specific histone 3 (CENH3) variant in plants. We show that CENH3 of Arabidopsis thaliana is phosphorylated in vitro by Aurora3, predominantly at serine 65. Interaction of Aurora3 and CENH3 was found by immunoprecipitation (IP) in A. thaliana and by bimolecular fluorescence complementation. Western blotting with an anti-CENH3 pS65 antibody showed that CENH3 pS65 is more abundant in flower buds than elsewhere in the plant. Substitution of serine 65 by either alanine or aspartic acid resulted in a range of phenotypic abnormalities, especially in reproductive tissues. We conclude that Aurora3 phosphorylates CENH3 at S65 and that this post-translational modification is required for the proper development of the floral meristem.

Variation in Recombination Rate Is Shaped by Domestication and Environmental Conditions in Barley.

Meiotic recombination generates genetic diversity upon which selection can act. Recombination rates are highly variable between species, populations, individuals, sexes, chromosomes, and chromosomal regions. The underlying mechanisms are controlled at the genetic and epigenetic level and show plasticity toward the environment. Environmental plasticity may be divided into short- and long-term responses. We estimated recombination rates in natural populations of wild barley and domesticated landraces using a population genetics approach. We analyzed recombination landscapes in wild barley and domesticated landraces at high resolution. In wild barley, high recombination rates are found in more interstitial chromosome regions in contrast to distal chromosome regions in domesticated barley. Among subpopulations of wild barley, natural variation in effective recombination rate is correlated with temperature, isothermality, and solar radiation in a nonlinear manner. A positive linear correlation was found between effective recombination rate and annual precipitation. We discuss our findings with respect to how the environment might shape effective recombination rates in natural populations. Higher recombination rates in wild barley populations subjected to specific environmental conditions could be a means to maintain fitness in a strictly inbreeding species.

Tackling Plant Meiosis: From Model Research to Crop Improvement.

Genetic engineering and traditional plant breeding, which harnesses the natural genetic variation that arises during meiosis, will have key roles to improve crop varieties and thus deliver Food Security in the future. Meiosis, a specialized cell division producing haploid gametes to maintain somatic diploidy following their fusion, assures genetic variation by regulated genetic exchange through homologous recombination. However, meiotic recombination events are restricted in their total number and their distribution along chromosomes limiting allelic variations in breeding programs. Thus, modifying the number and distribution of meiotic recombination events has great potential to improve and accelerate plant breeding. In recent years much progress has been made in understanding meiotic progression and recombination in plants. Many genes and factors involved in these processes have been identified primarily in Arabidopsis thaliana but also more recently in crops such as Brassica, rice, barley, maize, or wheat. These advances put researchers in the position to translate acquired knowledge to various crops likely improving and accelerating breeding programs. However, although fundamental aspects of meiotic progression and recombination are conserved between species, differences in genome size and organization (due to repetitive DNA content and ploidy level) exist, particularly among plants, that likely account for differences in meiotic progression and recombination patterns found between species. Thus, tools and approaches are needed to better understand differences and similarities in meiotic progression and recombination among plants, to study fundamental aspects of meiosis in a variety of plants including crops and non-model species, and to transfer knowledge into crop species. In this article, we provide an overview of tools and approaches available to study plant meiosis, highlight new techniques, give examples of areas of future research and review distinct aspects of meiosis in non-model species.

Identification of ASYNAPTIC4, a Component of the Meiotic Chromosome Axis.

During the leptotene stage of prophase I of meiosis, chromatids become organized into a linear looped array via a protein axis that forms along the loop bases. Establishment of the axis is essential for the subsequent synapsis of the homologous chromosome pairs and the progression of recombination to form genetic crossovers. Here, we describe ASYNAPTIC4 (ASY4), a meiotic axis protein in Arabidopsis (Arabidopsis thaliana). ASY4 is a small coiled-coil protein that exhibits limited sequence similarity with the carboxyl-terminal region of the axis protein ASY3. We used enhanced yellow fluorescent protein-tagged ASY4 to show that ASY4 localizes to the chromosome axis throughout prophase I. Bimolecular fluorescence complementation revealed that ASY4 interacts with ASY1 and ASY3, and yeast two-hybrid analysis confirmed a direct interaction between ASY4 and ASY3. Mutants lacking full-length ASY4 exhibited defective axis formation and were unable to complete synapsis. Although the initiation of recombination appeared to be unaffected in the asy4 mutant, the number of crossovers was reduced significantly, and crossovers tended to group in the distal parts of the chromosomes. We conclude that ASY4 is required for normal axis and crossover formation. Furthermore, our data suggest that ASY3/ASY4 are the functional homologs of the mammalian SYCP2/SYCP3 axial components.

Affinity proteomics reveals extensive phosphorylation of the Brassica chromosome axis protein ASY1 and a network of associated proteins at prophase I of meiosis.

During meiosis, the formation of crossovers (COs) generates genetic variation and provides physical links that are essential for accurate chromosome segregation. COs occur in the context of a proteinaceous chromosome axis. The transcriptomes and proteomes of anthers and meiocytes comprise several thousand genes and proteins, but because of the level of complexity relatively few have been functionally characterized. Our understanding of the physical and functional interactions between meiotic proteins is also limited. Here we use affinity proteomics to analyse the proteins that are associated with the meiotic chromosome axis protein, ASY1, in Brassica oleracea anthers and meiocytes. We show that during prophase I ASY1 and its interacting partner, ASY3, are extensively phosphorylated, and we precisely assign phosphorylation sites. We identify 589 proteins that co-immunoprecipitate with ASY1. These correspond to 492 Arabidopsis orthologues, over 90% of which form a coherent protein-protein interaction (PPI) network containing known and candidate meiotic proteins, including proteins more usually associated with other cellular processes such as DNA replication and proteolysis. Mutant analysis confirms that affinity proteomics is a viable strategy for revealing previously unknown meiotic proteins, and we show how the PPI network can be used to prioritise candidates for analysis. Finally, we identify another axis-associated protein with a role in meiotic recombination. Data are available via ProteomeXchange with identifier PXD006042.