Immunodetection of tubulin and centromeric histone H3 (CENH3) proteins in Glycine species.

BACKGROUND: The centromeres appear as primary constrictions on monocentric metaphase chromosomes; where sister chromatids are held together and assemble the proteinaceous kitechore complex at which microtubule proteins attach during nuclear divisions for pulling sister chromatids to opposite cell poles. The movement of chromosomes is usually governed by structural proteins that are either species-specific or highly conserved, such as the centromere-specific histone H3 (CENH3) and tubulin proteins, respectively. METHODS AND RESULTS: We aimed to detect these proteins across eight different Glycine species by an immunofluorescence assay using specific antibodies. Furthermore, with the α-tubulin antibody we traced the dynamics of microtubules during the mitotic cell cycle in Glycine max. With two-color immunofluorescence staining, we showed that both proteins interact during nuclear division. CONCLUSIONS: Finally, we proved that in different diploid and tetraploid Glycine species CENH3 can be detected in functional centromeres with spatial proximity of microtubule proteins.

Centromere sequence-independent but biased loading of subgenome-specific CENH3 variants in allopolyploid Arabidopsis suecica.

Centromeric nucleosomes are determined by the replacement of the canonical histone H3 with the centromere-specific histone H3 (CENH3) variant. Little is known about the centromere organization in allopolyploid species where different subgenome-specific CENH3s and subgenome-specific centromeric sequences coexist. Here, we analyzed the transcription and centromeric localization of subgenome-specific CENH3 variants in the allopolyploid species Arabidopsis suecica. Synthetic A. thaliana x A. arenosa hybrids were generated and analyzed to mimic the early evolution of A. suecica. Our expression analyses indicated that CENH3 has generally higher expression levels in A. arenosa compared to A. thaliana, and this pattern persists in the hybrids. We also demonstrated that despite a different centromere DNA composition, the centromeres of both subgenomes incorporate CENH3 encoded by both subgenomes, but with a positive bias towards the A. arenosa-type CENH3. The intermingled arrangement of both CENH3 variants demonstrates centromere plasticity and may be an evolutionary adaption to handle more than one CENH3 variant in the process of allopolyploidization.

The Chaperone NASP Contributes to De Novo Deposition of the Centromeric Histone Variant CENH3 in Arabidopsis Early Embryogenesis.

The centromere is an essential chromosome region where the kinetochore is formed to control equal chromosome distribution during cell division. The centromere-specific histone H3 variant CENH3 (also called CENP-A) is a prerequisite for the kinetochore formation. Since CENH3 evolves rapidly, associated factors, including histone chaperones mediating the deposition of CENH3 on the centromere, are thought to act through species-specific amino-acid sequences. The functions and interaction networks of CENH3 and histone chaperons have been well-characterized in animals and yeasts. However, molecular mechanisms involved in recognition and deposition of CENH3 are still unclear in plants. Here, we used a swapping strategy between domains of CENH3 of Arabidopsis thaliana and the liverwort Marchantia polymorpha to identify specific regions of CENH3 involved in targeting the centromeres and interacting with the general histone H3 chaperone, NASP (nuclear autoantigenic sperm protein). CENH3's LoopN-α1 region was necessary and sufficient for the centromere targeting in cooperation with the α2 region and was involved in interaction with NASP in cooperation with αN, suggesting a species-specific CENH3 recognition. In addition, by generating an Arabidopsis nasp knockout mutant in the background of a fully fertile GFP-CENH3/cenh3-1 line, we found that NASP was implicated for de novo CENH3 deposition after fertilization and thus for early embryo development. Our results imply that the NASP mediates the supply of CENH3 in the context of the rapidly evolving centromere identity in land plants.

Celine, a long interspersed nuclear element retrotransposon, colonizes in the centromeres of poplar chromosomes.

Centromeres in most multicellular eukaryotes are composed of long arrays of repetitive DNA sequences. Interestingly, several transposable elements, including the well-known long terminal repeat (LTR) retrotransposon CRM (centromeric retrotransposon of maize), were found to be enriched in functional centromeres marked by the centromeric histone H3 (CENH3). Here we report a centromeric long interspersed nuclear element (LINE), Celine, in Populus species. Celine has colonized preferentially in the CENH3-associated chromatin of every poplar chromosome, with 84% of the Celine elements localized in the CENH3-binding domains. By contrast, only 51% of the CRM elements were bound to CENH3 domains in Populus trichocarpa. These results suggest different centromere targeting mechanisms employed by Celine and CRM elements. Nevertheless, the high target specificity seems to be detrimental to further amplification of the Celine elements, leading to a shorter life span and patchy distribution among plant species compared to the CRM elements. Using a phylogenetically guided approach we were able to identify Celine-like LINE elements in tea plant (Camellia sinensis) and green ash tree (Fraxinus pennsylvanica). The centromeric localization of these Celine-like LINEs was confirmed in both species. We demonstrate that the centromere targeting property of Celine-like LINEs is of primitive origin and has been conserved among distantly related plant species.

Three near-complete genome assemblies reveal substantial centromere dynamics from diploid to tetraploid in Brachypodium genus.

BACKGROUND: Centromeres are critical for maintaining genomic stability in eukaryotes, and their turnover shapes genome architectures and drives karyotype evolution. However, the co-evolution of centromeres from different species in allopolyploids over millions of years remains largely unknown. RESULTS: Here, we generate three near-complete genome assemblies, a tetraploid Brachypodium hybridum and its two diploid ancestors, Brachypodium distachyon and Brachypodium stacei. We detect high degrees of sequence, structural, and epigenetic variations of centromeres at base-pair resolution between closely related Brachypodium genomes, indicating the appearance and accumulation of species-specific centromere repeats from a common origin during evolution. We also find that centromere homogenization is accompanied by local satellite repeats bursting and retrotransposon purging, and the frequency of retrotransposon invasions drives the degree of interspecies centromere diversification. We further investigate the dynamics of centromeres during alloploidization process, and find that dramatic genetics and epigenetics architecture variations are associated with the turnover of centromeres between homologous chromosomal pairs from diploid to tetraploid. Additionally, our pangenomes analysis reveals the ongoing variations of satellite repeats and stable evolutionary homeostasis within centromeres among individuals of each Brachypodium genome with different polyploidy levels. CONCLUSIONS: Our results provide unprecedented information on the genomic, epigenomic, and functional diversity of highly repetitive DNA between closely related species and their allopolyploid genomes at both coarse and fine scale.

KNL1 and NDC80 represent new universal markers for the detection of functional centromeres in plants.

Centromere is the chromosomal site of kinetochore assembly and microtubule attachment for chromosome segregation. Given its importance, markers that allow specific labeling of centromeric chromatin throughout the cell cycle and across all chromosome types are sought for facilitating various centromere studies. Antibodies against the N-terminal region of CENH3 are commonly used for this purpose, since CENH3 is the near-universal marker of functional centromeres. However, because the N-terminal region of CENH3 is highly variable among plant species, antibodies directed against this region usually function only in a small group of closely related species. As a more versatile alternative, we present here antibodies targeted to the conserved domains of two outer kinetochore proteins, KNL1 and NDC80. Sequence comparison of these domains across more than 350 plant species revealed a high degree of conservation, particularly within a six amino acid motif, FFGPVS in KNL1, suggesting that both antibodies would function in a wide range of plant species. This assumption was confirmed by immunolabeling experiments in angiosperm (monocot and dicot) and gymnosperm species, including those with mono-, holo-, and meta-polycentric chromosomes. In addition to centromere labeling on condensed chromosomes during cell division, both antibodies detected the corresponding regions in the interphase nuclei of most species tested. These results demonstrated that KNL1 and NDC80 are better suited for immunolabeling centromeres than CENH3, because antibodies against these proteins offer incomparably greater versatility across different plant species which is particularly convenient for studying the organization and function of the centromere in non-model species.

Chromosome-level changes and genome elimination by manipulation of CENH3 in carrot (Daucus carota).

Hybrid cultivars are valuable in many crop species due to their high yield, uniformity, and other desirable traits. Doubled haploids, which have two identical sets of chromosomes, are valuable for hybrid breeding because they can be produced in one generation, in comparison to the multigenerational process typically used to produce inbred parents for hybrid production. One method to produce haploid plants is manipulation of centromeric histone H3 (CENH3). This method of producing haploids has so far been successful in Arabidopsis, maize (Zea mays), and wheat (Triticum aestivum). Here we describe modification of CENH3 in carrot (Daucus carota) to test for the ability of these modifications to induce uniparental genome elimination, which is the basis for haploid induction. Base editing was used to make cenh3 mutant plants with amino acid substitutions in the region of CENH3 encoding the histone fold domain. These cenh3 mutant plants were then outcrossed with CENH3 wild-type plants. Using PCR-based genotyping assays, we identified two candidates for genome elimination. One candidate was classified as a putative aneuploid plant in which chromosome 7 is in a single copy state. The other candidate was characterized as a putative tetraploid that was likely haploid during its genesis. Our results suggest that this putative tetraploid inherited all of its chromosomes from the CENH3 wild-type parent and that the genome of the cenh3 mutant plant was lost. This study provides evidence that modification of CENH3 in carrot has the potential to induce genome elimination and ploidy changes in carrot.

Pan-centromere reveals widespread centromere repositioning of soybean genomes.

Centromere repositioning refers to a de novo centromere formation at another chromosomal position without sequence rearrangement. This phenomenon was frequently encountered in both mammalian and plant species and has been implicated in genome evolution and speciation. To understand the dynamic of centromeres on soybean genome, we performed the pan-centromere analysis using CENH3-ChIP-seq data from 27 soybean accessions, including 3 wild soybeans, 9 landraces, and 15 cultivars. Building upon the previous discovery of three centromere satellites in soybean, we have identified two additional centromere satellites that specifically associate with chromosome 1. These satellites reveal significant rearrangements in the centromere structures of chromosome 1 across different accessions, consequently impacting the localization of CENH3. By comparative analysis, we reported a high frequency of centromere repositioning on 14 out of 20 chromosomes. Most newly emerging centromeres formed in close proximity to the native centromeres and some newly emerging centromeres were apparently shared in distantly related accessions, suggesting their emergence is independent. Furthermore, we crossed two accessions with mismatched centromeres to investigate how centromere positions would be influenced in hybrid genetic backgrounds. We found that a significant proportion of centromeres in the S9 generation undergo changes in size and position compared to their parental counterparts. Centromeres preferred to locate at satellites to maintain a stable state, highlighting a significant role of centromere satellites in centromere organization. Taken together, these results revealed extensive centromere repositioning in soybean genome and highlighted how important centromere satellites are in constraining centromere positions and supporting centromere function.

Meiotic segregation and post-meiotic drive of the Festuca pratensis B chromosome.

In many species, the transmission of B chromosomes (Bs) does not follow the Mendelian laws of equal segregation and independent assortment. This deviation results in transmission rates of Bs higher than 0.5, a process known as "chromosome drive". Here, we studied the behavior of the 103 Mbp-large B chromosome of Festuca pratensis during all meiotic and mitotic stages of microsporogenesis. Mostly, the B chromosome of F. pratensis segregates during meiosis like standard A chromosomes (As). In some cases, the B passes through meiosis in a non-Mendelian segregation leading to their accumulation already in meiosis. However, a true drive of the B happens during the first pollen mitosis, by which the B preferentially migrates to the generative nucleus. During second pollen mitosis, B divides equally between the two sperms. Despite some differences in the frequency of drive between individuals with different numbers of Bs, at least 82% of drive was observed. Flow cytometry-based quantification of B-containing sperm nuclei agrees with the FISH data.

Simultaneous EYFP-CENH3/H2B-DsRed Expression Is Impaired Differentially in Meristematic and Differentiated Nuclei of Arabidopsis Double Transformants.

Fluorescence live-cell microscopy is important in cell biology to perform artifact-free investigations. To analyze the dynamics of chromatin and centromeres at different stages of the cell cycle in nuclei and chromosomes, we performed simultaneous EYFP-CENH3/H2B-DsRed and single H2B-YFP transformations in Arabidopsis wild-type and cohesin T-DNA mutants. All constructs were under the control of the strong CaMV 35S promoter. While a strong silencing of fluorescence expression occurred differently in leaf and root tissues in the double transformants, nearly all single-transformed wild-type and most mutant cells showed H2B-YFP fluorescence. It seems that for an efficient co-expression of two fluorescence proteins, endogenous promoters and terminators should be used.

Centromere Plasticity With Evolutionary Conservation and Divergence Uncovered by Wheat 10+ Genomes.

Centromeres (CEN) are the chromosomal regions that play a crucial role in maintaining genomic stability. The underlying highly repetitive DNA sequences can evolve quickly in most eukaryotes, and promote karyotype evolution. Despite their variability, it is not fully understood how these widely variable sequences ensure the homeostasis of centromere function. In this study, we investigated the genetics and epigenetics of CEN in a population of wheat lines from global breeding programs. We captured a high degree of sequences, positioning, and epigenetic variations in the large and complex wheat CEN. We found that most CENH3-associated repeats are Cereba element of retrotransposons and exhibit phylogenetic homogenization across different wheat lines, but the less-associated repeat sequences diverge on their own way in each wheat line, implying specific mechanisms for selecting certain repeat types as functional core CEN. Furthermore, we observed that CENH3 nucleosome structures display looser wrapping of DNA termini on complex centromeric repeats, including the repositioned CEN. We also found that strict CENH3 nucleosome positioning and intrinsic DNA features play a role in determining centromere identity among different lines. Specific non-B form DNAs were substantially associated with CENH3 nucleosomes for the repositioned centromeres. These findings suggest that multiple mechanisms were involved in the adaptation of CENH3 nucleosomes that can stabilize CEN. Ultimately, we proposed a remarkable epigenetic plasticity of centromere chromatin within the diverse genomic context, and the high robustness is crucial for maintaining centromere function and genome stability in wheat 10+ lines as a result of past breeding selections.

Recent Advances in Engineering of In Vivo Haploid Induction Systems.

Doubled haploid (DH) technology is an important approach to accelerate genetic gain via a shortened breeding cycle, which relies on the ability to generate haploid cells that develop into haploids or doubled haploid embryos and plants. Both in vitro and in vivo (in seed) methods can be used for haploid production. In vitro culture of gametophytes (microspores and megaspores) or their surrounding floral tissues or organs (anthers, ovaries, or ovules) has generated haploid plants in wheat, rice, cucumber, tomato, and many other crops. In vivo methods utilize pollen irradiation or wide crossing or in certain species leverage genetic mutant haploid inducer lines. Haploid inducers were widespread in corn and barley, and recent cloning of the inducer genes and identification of the causal mutations in corn have led to the establishment of in vivo haploid inducer systems via genome editing of orthologous genes in more diverse species. Further combination of DH and genome editing technology led to the development of novel breeding technologies such as HI-EDIT™. In this chapter, we will review in vivo haploid induction and new breeding technologies that combine haploid induction and genome editing.

Characterization of centromeric DNA of Gossypium anomalum reveals sequence-independent enrichment dynamics of centromeric repeats.

Centromeres in eukaryotes are composed of highly repetitive DNAs, which evolve rapidly and are thought to achieve a favorable structure in mature centromeres. However, how the centromeric repeat evolves into an adaptive structure is largely unknown. We characterized the centromeric sequences of Gossypium anomalum through chromatin immunoprecipitation against CENH3 antibodies. We revealed that the G. anomalum centromeres contained only retrotransposon-like repeats but were depleted in long arrays of satellites. These retrotransposon-like centromeric repeats were present in the African-Asian and Australian lineage species, suggesting that they might have arisen in the common ancestor of these diploid species. Intriguingly, we observed a substantial increase and decrease in copy numbers among African-Asian and Australian lineages, respectively, for the retrotransposon-derived centromeric repeats without apparent structure or sequence variation in cotton. This result indicates that the sequence content is not a decisive aspect of the adaptive evolution of centromeric repeats or at least retrotransposon-like centromeric repeats. In addition, two active genes with potential roles in gametogenesis or flowering were identified in CENH3 nucleosome-binding regions. Our results provide new insights into the constitution of centromeric repetitive DNA and the adaptive evolution of centromeric repeats in plants.

Accelerated Breeding for Helianthus annuus (Sunflower) through Doubled Haploidy: An Insight on Past and Future Prospects in the Era of Genome Editing.

The aim of any breeding process is to fully express the targeted, superior/desirable parent characteristic in the progeny. Hybrids are often used in this dynamic, and complex process for which homozygous parents-which may require up to eight generations of back crossing and selection-are required. Doubled haploid (DH) technologies can facilitate the production of true breeding lines faster and in a more efficient manner than the traditional back crossing and selection strategies. Sunflower is the third most important oilseed crop in the world and has no available double haploid induction procedure/technique that can be efficiently used in breeding programs. A reproducible and efficient doubled haploid induction method would be a valuable tool in accelerating the breeding of new elite sunflower varieties. Although several attempts have been made, the establishment of a sunflower doubled haploid induction protocol has remained a challenge owing recalcitrance to in vitro culture regeneration. Approaches for haploid development in other crops are often cultivar specific, difficult to reproduce, and rely on available tissue culture protocols-which on their own are also cultivar and/or species specific. As an out-crossing crop, the lack of a double haploid system limits sunflower breeding and associated improvement processes, thereby delaying new hybrid and trait developments. Significant molecular advances targeting genes, such as the centromeric histone 3 (CenH3) and Matrilineal (MTL) gene with CRISPR/Cas9, and the successful use of viral vectors for the delivery of CRISPR/Cas9 components into plant cells eliminating the in vitro culture bottleneck, have the potential to improve double haploid technology in sunflower. In this review, the different strategies, their challenges, and opportunities for achieving doubled haploids in sunflower are explored.

Super-resolution microscopy reveals the number and distribution of topoisomerase IIα and CENH3 molecules within barley metaphase chromosomes.

Topoisomerase IIα (Topo IIα) and the centromere-specific histone H3 variant CENH3 are key proteins involved in chromatin condensation and centromere determination, respectively. Consequently, they are required for proper chromosome segregation during cell divisions. We combined two super-resolution techniques, structured illumination microscopy (SIM) to co-localize Topo IIα and CENH3, and photoactivated localization microscopy (PALM) to determine their molecule numbers in barley metaphase chromosomes. We detected a dispersed Topo IIα distribution along chromosome arms but an accumulation at centromeres, telomeres, and nucleolus-organizing regions. With a precision of 10-50 nm, we counted ~ 20,000-40,000 Topo IIα molecules per chromosome, 28% of them within the (peri)centromere. With similar precision, we identified ~13,500 CENH3 molecules per centromere where Topo IIα proteins and CENH3-containing chromatin intermingle. In short, we demonstrate PALM as a useful method to count and localize single molecules with high precision within chromosomes. The ultrastructural distribution and the detected amount of Topo IIα and CENH3 are instrumental for a better understanding of their functions during chromatin condensation and centromere determination.

Heat stress promotes haploid formation during CENH3-mediated genome elimination in Arabidopsis.

Impaired activity of centromeric histone CENH3 causes inaccurate chromosome segregation and in crosses between the Arabidopsis recombinant CENH3 mutant GFP-tailswap and CENH3G83E with wild-type pollen it results in chromosome loss with the formation of haploids. This genome elimination in the zygote and embryo is not absolute as also aneuploid and diploid progeny is formed. Here, we report that a temporal and moderate heat stress during fertilization and early embryogenesis shifts the ratio in favour of haploid progeny in CENH3 mutant lines. Micronuclei formation, a proxy for genome elimination, was similar in control and heat-treated flowers, indicating that heat-induced seed abortion occurred at a late stage during the development of the seed. In the seeds derived from heat-treated crosses, the endosperm did not cellularize and many seeds aborted. Haploid seeds were formed, however, resulting in increased frequencies of haploids in CENH3-mediated genome elimination crosses performed under heat stress. Therefore, heat stress application is a selective force during genome elimination that promotes haploid formation and may be used to improve the development and efficacy of in vivo haploid induction systems.

Non-Rabl chromosome organization in endoreduplicated nuclei of barley embryo and endosperm tissues.

Rabl organization is a type of interphase chromosome arrangement with centromeres and telomeres clustering at opposite nuclear poles. Here, we analyzed nuclear morphology and chromosome organization in cycling and endoreduplicated nuclei isolated from embryo and endosperm tissues of developing barley seeds. We show that endoreduplicated nuclei have an irregular shape, less sister chromatid cohesion at 5S rDNA loci, and a reduced amount of centromeric histone CENH3. While the chromosomes of the embryo and endosperm nuclei are initially organized in Rabl configuration, the centromeres and telomeres are intermingled within the nuclear space in the endoreduplicated nuclei with an increasing endoreduplication level. Such a loss of chromosome organization suggests that Rabl configuration is introduced and further reinforced by mitotic divisions in barley cell nuclei in a tissue- and seed age-dependent manner.

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.

Rapid creation of CENH3-mediated haploid induction lines using a cytosine base editor (CBE).

Haploid induction (HI) can create true-breeding lines in a single generation, which can significantly accelerates the breeding process. In recent years, scientists have developed a variety of new techniques to induce haploids through manipulation of CENH3, a variant of the centromere-specific histone H3. One alternative approach is based on CENH3 point mutations derived from EMS/TILLING, which is not lethal and yet is responsible for inducing haploids. However, most residues have been obtained by EMS mutagenesis over a long period of time. Recently, a new approach called 'base editing' was developed for plants. Here, we report a new method that uses a cytosine base editor (CBE) to create a point mutation of CENH3 as a haploid induction line, which substitutes adenine (A) for guanine (G). As proof of the extreme simplicity of this approach to create haploid-induced lines, we identified an L130F substitution within the histone fold domain in Arabidopsis thaliana. Subsequently, we tested the haploid-inducing potential of homozygous L130F plants by pollinating them with Col-0, and obtained 2.9% paternal haploid plants. In brief, our innovative technology provides a new perspective for the promotion of CENH3-mediated haploid induction in crops, and also provides a variety of options for breeders. Such conserved point mutations as L130F could be developed into a general instrument for haploid induction in a wide range of plant species. Extending these systems would represent a major advance over haploid production.

Haploid induction by nanobody-targeted ubiquitin-proteasome-based degradation of EYFP-tagged CENH3 in Arabidopsis thaliana.

The generation of haploid plants accelerates the crop breeding process. One of the haploidization strategies is based on the genetic manipulation of endogenous centromere-specific histone 3 (CENH3). To extend the haploidization toolbox, we tested whether targeted in vivo degradation of CENH3 protein can be harnessed to generate haploids in Arabidopsis thaliana. We show that a recombinant anti-GFP nanobody fused to either heterologous F-box (NSlmb) or SPOP/BTB ligase proteins can recognize maternally derived enhanced yellow fluorescent protein (EYFP)-tagged CENH3 in planta and make it accessible for the ubiquitin-proteasome pathway. Outcrossing of the genomic CENH3-EYFP-complemented cenh3.1 mother with plants expressing the GFP-nanobody-targeted E3 ubiquitin ligase resulted in a haploid frequency of up to 7.6% in pooled F1 seeds. EYFP-CENH3 degradation occurred independently in embryo and endosperm cells. In reciprocal crosses, no haploid induction occurred. We propose that the uniparental degradation of EYFP-fused genomic CENH3 during early embryogenesis leads to a decrease in its level at centromeres and subsequently weakens the centromeres. The male-derived wild type CENH3 containing centromere outcompetes the CENH3-EYFP depleted centromere. Consequently, maternal chromosomes undergo elimination, resulting in haploids.