Surface structures consisting of chromatin fibers in isolated barley (Hordeum vulgare) chromosomes revealed by helium ion microscopy.

The chromosome compaction of chromatin fibers results in the formation of the nucleosome, which consists of a DNA unit coiled around a core of histone molecules associated with linker histone. The compaction of chromatin fibers has been a topic of controversy since the discovery of chromosomes in the 19th century. Although chromatin fibers were first identified using electron microscopy, the chromatin fibers on the surface of chromosome structures in plants remain unclear due to shrinking and breaking caused by prior chromosome isolation or preparation with alcohol and acid fixation, and critical point drying occurred into dehydration and denatured chromosomal proteins. This study aimed to develop a high-quality procedure for the isolation and preparation of plant chromosomes, maintaining the native chromosome structure, to elucidate the organization of chromatin fibers on the surface of plant chromosomes by electron microscopy. A simple technique to isolate intact barley (Hordeum vulgare) chromosomes with a high yield was developed, allowing chromosomes to be observed with a high-resolution scanning ion microscopy and helium ion microscopy (HIM) imaging technology, based on a scanning helium ion beam. HIM images from the surface chromatin fibers were analyzed to determine the size and alignment of the chromatin fibers. The unit size of the chromatin fibers was 11.6 ± 3.5 nm and was closely aligned to the chromatin network model. Our findings indicate that compacting the surface structure of barley via a chromatin network and observation via HIM are powerful tools for investigating the structure of chromatin.

Higher-Order Structure of Human Chromosomes Observed by Electron Diffraction and Electron Tomography.

It is well known that two DNA molecules are wrapped around histone octamers and folded together to form a single chromosome. However, the nucleosome fiber folding within a chromosome remains an enigma, and the higher-order structure of chromosomes also is not understood. In this study, we employed electron diffraction which provides a noninvasive analysis to characterize the internal structure of chromosomes. The results revealed the presence of structures with 100­200 nm periodic features directionally perpendicular to the chromosome axis in unlabeled isolated human chromosomes. We also visualized the 100­200 nm periodic features perpendicular to the chromosome axis in an isolated chromosome whose DNA molecules were specifically labeled with OsO4 using electron tomography in 300 keV and 1 MeV transmission electron microscopes.

Physical clustering of FLC alleles during Polycomb-mediated epigenetic silencing in vernalization.

Vernalization, the promotion of flowering by cold, involves Polycomb-mediated epigenetic silencing of FLOWERING LOCUS C (FLC). Cold progressively promotes cell-autonomous switching to a silenced state. Here, we used live-cell imaging of FLC-lacO to monitor changes in nuclear organization during vernalization. FLC-lacO alleles physically cluster during the cold and generally remain so after plants are returned to warm. Clustering is dependent on the Polycomb trans-factors necessary for establishment of the FLC silenced state but not on LIKE HETEROCHROMATIN PROTEIN 1, which functions to maintain silencing. These data support the view that physical clustering may be a common feature of Polycomb-mediated epigenetic switching mechanisms.

Epigenetic Distribution of Recombinant Plant Chromosome Fragments in a Human-Arabidopsis Hybrid Cell Line.

Methylation systems have been conserved during the divergence of plants and animals, although they are regulated by different pathways and enzymes. However, studies on the interactions of the epigenomes among evolutionarily distant organisms are lacking. To address this, we studied the epigenetic modification and gene expression of plant chromosome fragments (~30 Mb) in a human-Arabidopsis hybrid cell line. The whole-genome bisulfite sequencing results demonstrated that recombinant Arabidopsis DNA could retain its plant CG methylation levels even without functional plant methyltransferases, indicating that plant DNA methylation states can be maintained even in a different genomic background. The differential methylation analysis showed that the Arabidopsis DNA was undermethylated in the centromeric region and repetitive elements. Several Arabidopsis genes were still expressed, whereas the expression patterns were not related to the gene function. We concluded that the plant DNA did not maintain the original plant epigenomic landscapes and was under the control of the human genome. This study showed how two diverging genomes can coexist and provided insights into epigenetic modifications and their impact on the regulation of gene expressions between plant and animal genomes.

Global epigenetic changes of histone modification under environmental stresses in rice root.

Abiotic stresses are non-living factors with negative morphological and physiological effects on living organisms. Substantial evidence exists that gene expression changes during plant cell growth are regulated by chromatin reconfiguration and histone modification. Several types of histone modifications are dramatically transformed in stress-responsive gene regions under drought stress conditions. Environmental stresses also cause the root apical meristem (RAM) region to decelerate root growth. In this study, we investigated how quantitative changes in epigenetic markers in this region influence rice morphology and physiology. Both iron and salinity treatments changed the epigenetic landscape from euchromatic to heterochromatic according to heterochromatin (H3K9me2) and euchromatin (H3K4me) markers, especially in the proximal meristem region. Moreover, supplementation with external abscisic acid (ABA) was able to mimic the effect of environmental stresses on global epigenetic changes. In contrast, the addition of external auxin (IAA) to rice under saline conditions affected heterochromatin formation without influencing euchromatin transformation. Chromatin dynamics is therefore believed to be directly connected to plant growth regulator signaling. We discuss insights into the role of plant growth regulators: ABA and IAA, peroxide signaling, and their effects on the global epigenetic change of histone modification under abiotic stresses.

Molecular insights into the non-recombining nature of the spinach male-determining region.

Spinach (Spinacia oleracea L.) is a dioecious plant with male heterogametic sex determination and homomorphic sex chromosomes (XY). The dioecism is utilized for producing commercial hybrid seeds, and hence understanding the molecular-genetic basis of the species' sex determining locus is an important issue for spinach breeding. In this study, seven dominant DNA markers were shown to completely co-segregate with the male-determining gene in segregating spinach populations comprising > 1500 plants. In addition, these seven dominant DNA markers were completely associated with the male-determining gene in over 100 spinach germplasm accessions and cultivars. These observations suggest that, in spinach, a Y-chromosomal region around the male-determining locus does not (or almost not) recombine with a counterpart region on the X chromosome. Using five of the seven DNA markers, five bacterial artificial chromosome (BAC) clone contigs with a total length of approximately 690 kbp were constructed. Full sequencing of six representative BAC clones (total insert length 504 kbp) from the five contigs and a transcriptome analysis by RNA-seq revealed that the Y-chromosomal region around the male-determining locus contains large amounts of repetitive elements, suggesting that the region might be poor in gene content. Most of the repeats found in this region are novel Ty1-copia-like and its derivative elements that accumulate predominantly in heterochromatic regions. Our findings may provide valuable insight into spinach genome structure and clues for future research into the evolution of the sex determining locus.

Cell differentiation and development in Arabidopsis are associated with changes in histone dynamics at the single-cell level.

The mechanism whereby the same genome can give rise to different cell types with different gene expression profiles is a fundamental problem in biology. Chromatin organization and dynamics have been shown to vary with altered gene expression in different cultured animal cell types, but there is little evidence yet from whole organisms linking chromatin dynamics with development. Here, we used both fluorescence recovery after photobleaching and two-photon photoactivation to show that in stem cells from Arabidopsis thaliana roots the mobility of the core histone H2B, as judged by exchange dynamics, is lower than in the surrounding cells of the meristem. However, as cells progress from meristematic to fully differentiated, core histones again become less mobile and more strongly bound to chromatin. We show that these transitions are largely mediated by changes in histone acetylation. We further show that altering histone acetylation levels, either in a mutant or by drug treatment, alters both the histone mobility and markers of development and differentiation. We propose that plant stem cells have relatively inactive chromatin, but they keep the potential to divide and differentiate into more dynamic states, and that these states are at least in part determined by histone acetylation levels.

Chromosome interior observation by focused ion beam/scanning electron microscopy (FIB/SEM) using ionic liquid technique.

Attempts to elucidate chromosome structure have long remained elusive. Electron microscopy is useful for chromosome structure research because of its high resolution and magnification. However, biological samples such as chromosomes need to be subjected to various preparation steps, including dehydration, drying, and metal/carbon coating, which may induce shrinkage and artifacts. The ionic liquid technique has recently been developed and it enables sample preparation without dehydration, drying, or coating, providing a sample that is closer to the native condition. Concurrently, focused ion beam/scanning electron microscopy (FIB/SEM) has been developed, allowing the investigation and direct analysis of chromosome interiors. In this study, we investigated chromosome interiors by FIB/SEM using plant and human chromosomes prepared by the ionic liquid technique. As a result, two types of chromosomes, with and without cavities, were visualized, both for barley and human chromosomes prepared by critical point drying. However, chromosome interiors were revealed only as a solid structure, lacking cavities, when prepared by the ionic liquid technique. Our results suggest that the existence and size of cavities depend on the preparation procedures. We conclude that combination of the ionic liquid technique and FIB/SEM is a powerful tool for chromosome study.

Habitat diversification associated with urban development has a little effect on genetic structure in the annual native plant Commelina communis in an East Asian megacity.

Urban development greatly alters the natural and semi-natural habitats of native plants. Urbanisation results in a range of diverse habitats including remnant agricultural lands, urban parks, and roadside habitats. This habitat diversity often promotes trait divergence within urban areas. However, the mechanisms by which diverse urban habitats influence the population genetic structure of individual plant species remain poorly understood. We investigated the effects of urbanisation on genetic diversity and structure within 24 Commelina communis populations across diverse habitat types (rural agricultural land, urban agricultural land, urban park land, and urban roadsides) within the Kyoto-Osaka-Kobe megacity in Japan. We conducted multiplexed inter-simple sequence repeat genotyping to compare genetic diversity among populations in different habitats. We also examined the correlation between Nei's genetic distance and geographic and environmental distances and performed principal coordinate analysis (PCoA) to evaluate genetic differentiation among urban habitats. There were no significant differences in genetic diversity indices between urban and rural populations and among urban habitat types. Although we detected no isolation-by-distance structure in population pairs of the same habitat type and in those of different habitats, the difference in surrounding landscape facilitated genetic differentiation not only between urban and rural habitats but also between different urban habitats. PCoA revealed no clear genetic differentiation among rural and urban habitat populations. Our findings indicate that the establishment of diverse habitat types through urbanisation has no and little impact on genetic diversity and structure, respectively, in C. communis, likely due to its high selfing rate and ability to adapt to urban conditions.

Localization and quantitative distribution of a chromatin structural protein Topoisomerase II on plant chromosome using HVTEM and UHVTEM.

Topoisomerase II (TopoII) is an essential structural protein of the metaphase chromosome. It maintains the axial compaction of chromosomes during metaphase. It is localized at the axial region of chromosomes and accumulates at the centromeric region in metaphase chromosomes. However, little is known about TopoII localization and distribution in plant chromosomes, except for several publications. We used high voltage transmission electron microscopy (HVTEM) and ultra-high voltage transmission electron microscopy (UHVTEM) in conjunction with immunogold labeling and visualization techniques to detect TopoII and investigate its localization, alignment, and density on the barley chromosome at 1.4 nm scale. We found that HVTEM and UHVTEM combined with immunogold labeling is suitable for the detection of structural proteins, including a single molecule of TopoII. This is because the average size of the gold particles for TopoII visualization after silver enhancement is 8.9 ± 3.9 nm, which is well detected. We found that 31,005 TopoII molecules are distributed along the barley chromosomes in an unspecific pattern at the chromosome arms and accumulate specifically at the nucleolus organizer regions (NORs) and centromeric region. The TopoII density were 1.32-fold, 1.58-fold, and 1.36-fold at the terminal region, at the NORs, and the centromeric region, respectively. The findings of TopoII localization in this study support the multiple reported functions of TopoII in the barley metaphase chromosome.

Chromatin Immunostaining of Plant Nuclei.

Recent evidence has demonstrated that specific epigenetic changes are also related to plant growth and development. Immunostaining enables the detection and characterization of chromatin modification, e.g., histone H4 acetylation (H4K5ac), histone H3 methylation (H3K4me2 and H3K9me2), and DNA methylation (5mC) with unique and specific patterns in plant tissues. Here we describe experimental procedures to determine the histone H3 methylation (H3K4me2 and H3K9me2) patterns in 3D-chromatin in whole roots tissue and 2D-chromatin in single nuclei in rice. To analyze both iron and salinity treatments, we show how to test for changes to the epigenetic chromatin landscape using heterochromatin (H3K9me2) and euchromatin (H3K4me) markers for chromatin immunostaining, especially in the proximal meristem region. To elucidate the epigenetic impact of environmental stress and external plant growth regulators, we demonstrate how to apply a combination of salinity, auxin, and abscisic acid treatments. The results of these experiments provide insights into the epigenetic landscape during rice root growth and development.

CRISPR imaging reveals chromatin fluctuation at the centromere region related to cellular senescence.

The human genome is spatially and temporally organized in the nucleus as chromatin, and the dynamic structure of chromatin is closely related to genome functions. Cellular senescence characterized by an irreversible arrest of proliferation is accompanied by chromatin reorganisation in the nucleus during senescence. However, chromatin dynamics in chromatin reorganisation is poorly understood. Here, we report chromatin dynamics at the centromere region during senescence in cultured human cell lines using live imaging based on the clustered regularly interspaced short palindromic repeat/dCas9 system. The repetitive sequence at the centromere region, alpha-satellite DNA, was predominantly detected on chromosomes 1, 12, and 19. Centromeric chromatin formed irregular-shaped domains with high fluctuation in cells undergoing 5'-aza-2'-deoxycytidine-induced senescence. Our findings suggest that the increased fluctuation of the chromatin structure facilitates centromere disorganisation during cellular senescence.

Ultrastructure of Javaen barb fish Systomus orphoides Valenciennes, 1842 spermatozoa (Cypriniformes: Cyprinidae) by electron microscopes.

Javaen barb fish Systomus orphoides Valenciennes, 1842 (Cypriniformes: Cyprinidae) is a freshwater fish whose population is declining and threatened with extinction. In this study, the ultrastructure of spermatozoa of Javaen barb fish (S. orphoides) was studied using transmission and scanning electron microscopy. The spermatozoa of S. orphoides are relatively simple cells composed of a spherical head, a short midpiece, and a flagellum, as in most Cyprinidae. The ultrastructure is characterized by the absence of acrosome, the total length of spermatozoa is 27.16 ± 4.5 µm, and the head has spherical with a length of 1.84 ± 0.10 µm and width of 1.55 ± 0.15 µm containing a nucleus, midpiece region containing the proximal and distal centrioles and mitochondria. Two or three mitochondria surrounding the axoneme (with a 9 + 2 microtubular pattern). Ultrastructural analyses by SEM and TEM of Javaen barb fish spermatozoa cells are very consistent with those of Cyprinidae. This study provides the ultrastructure information of S. orphoides spermatozoa in the Cyprinidae family this research could be useful in increasing reproductive efficiency and further prevent the extinction of this species.

Higher-order structure of barley chromosomes observed by electron tomography.

The higher order structure of the metaphase chromosome has been an enigma for over a century and several different models have been presented based on results obtained by a variety of techniques. Some disagreements in the results between methods have possibly arisen from artifacts caused during sample preparation such as staining and dehydration. Therefore, we treated barley chromosomes with ionic liquid to minimize the effects of dehydration. We also observed chromosomes on a film with holes to keep pristine chromosome structure from being flattened as seen when placed on a continuous support film. A chromosome placed over a hole in a thin carbon film was mounted on a tomography holder, and its structure was observed in three dimensions (3D) using electron tomography. We found that there are periodic structures with 300-400 nm pitch along the axis in barley chromosomes. The pitch sizes are larger than those observed in human chromosomes.

Integration of cytogenetic and genetic linkage maps of Lotus japonicus, a model plant for legumes.

To construct a high-resolution pachytene chromosome map, we used the chromosome image analyzing system version 3 and fluorescence in situ hybridization. Two ribosomal RNA genes (45S rDNA and 5S rDNA), two major tandem repeat DNAs (LjTR1 and LjTR2), two major retroelements (LjRE1 and LjRE2), and 27 transformation-competent artificial chromosome clones were physically localized on Lotus japonicus (Miyakojima MG-20, 2n = 12) chromosomes. The distributions of heterochromatin and euchromatin along six chromosomes were compared based on the linkage map. Distortion between the recombination frequencies and physical chromosomal distance was recognized where the centromeric heterochromatic regions and constitutive heterochromatin are composed of the highest copy tandem repeat LjTR1 on the interstitial specific regions. Our study shows that the heterochromatin are composed of the specific repeated sequences, and the discrepancy between the recombination frequency and cytological information detected in L. japonicus chromosomes is due to the heterochromatin.

Water Spectral Patterns Reveals Similarities and Differences in Rice Germination and Induced Degenerated Callus Development.

In vivo monitoring of rice (Oryza sativa L.) seed germination and seedling growth under general conditions in closed Petri dishes containing agar base medium at room temperature (temperature = 24.5 ± 1 °C, relative humidity = 76 ± 7% (average ± standard deviation)), and induced degenerated callus formation with plant growth regulator, were performed using short-wavelength near-infrared spectroscopy and aquaphotomics over A period of 26 days. The results of spectral analysis suggest changes in water absorbances due to the production of common metabolites, as well as increases in biomass and the sizes of the samples. Quantitative models built to predict the day of the development provided better accuracy for rice seedlings growth compared to callus formation. Eight common water bands were identified as presenting prominent changes in the absorbance pattern. The water matrix of only rice seedlings showed three developmental stages: firstly expressing a predominantly weakly hydrogen-bonded state, then a more strongly hydrogen-bonded state, and then, again, a weakly hydrogen-bonded state at the end. In rice callus induction and proliferation, no similar change in water absorbance pattern was observed. The presented findings indicate the potential of aquaphotomics for the in vivo detection of degeneration in cell development.

Molecular organization of recombinant human-Arabidopsis chromosomes in hybrid cell lines.

Although plants and animals are evolutionarily distant, the structure and function of their chromosomes are largely conserved. This allowed the establishment of a human-Arabidopsis hybrid cell line in which a neo-chromosome was formed by insertion of segments of Arabidopsis chromosomes into human chromosome 15. We used this unique system to investigate how the introgressed part of a plant genome was maintained in human genetic background. The analysis of the neo-chromosome in 60- and 300-day-old cell cultures by next-generation sequencing and molecular cytogenetics suggested its origin by fusion of DNA fragments of different sizes from Arabidopsis chromosomes 2, 3, 4, and 5, which were randomly intermingled rather than joined end-to-end. The neo-chromosome harbored Arabidopsis centromeric repeats and terminal human telomeres. Arabidopsis centromere wasn't found to be functional. Most of the introgressed Arabidopsis DNA was eliminated during the culture, and the Arabidopsis genome in 300-day-old culture showed significant variation in copy number as compared with the copy number variation in the 60-day-old culture. Amplified Arabidopsis centromere DNA and satellite repeats were localized at particular loci and some fragments were inserted into various positions of human chromosome. Neo-chromosome reorganization and behavior in somatic cell hybrids between the plant and animal kingdoms are discussed.

Calcium ion significance on the maintenance of barley (Hordeum vulgare) chromosome compaction.

Cations, especially calcium ions (Ca2+), is one of the major factors responsible for the chromosome higher-order structure formation. The effects of cations on the human chromosomes have already been evaluated, however, whether the presence of similar effects on plant chromosomes has not been reported to date. Thus, in this study, we investigated the role of Ca2+ on the barley (Hordeum vulgare L.) chromosome structure. Barley chromosomes were isolated from the meristematic tissue within the germinated roots. The roots were subjected to enzymatic treatment, fixed, and drop on the cover glass to spread the chromosomes out. Some chromosomes were treated with BAPTA (1,2-Bis(2-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid) to chelate Ca2+. Chromosome samples were then observed by fluorescence microscopy and scanning electron microscopy (SEM). The disperse structure of the chromosome was observed after BAPTA treatment. Chromosomes showed less condensed structure due to Ca2+ chelation. The high-resolution of SEM provided a more detailed visualization of chromosome ultrastructure under different calcium ion conditions. This study revealed the calcium ion effect on chromosome structure is important regardless of the organisms, suggesting a similar mechanism of chromosome condensation through humans and plants.

Recent advances in rice genome and chromosome structure research by fluorescence in situ hybridization (FISH).

Fluorescence in situ hybridization (FISH) is an effective method for the physical mapping of genes and repetitive DNA sequences on chromosomes. Physical mapping of unique nucleotide sequences on specific rice chromosome regions was performed using a combination of chromosome identification and highly sensitive FISH. Increases in the detection sensitivity of smaller DNA sequences and improvements in spatial resolution have ushered in a new phase in FISH technology. Thus, it is now possible to perform in situ hybridization on somatic chromosomes, pachytene chromosomes, and even on extended DNA fibers (EDFs). Pachytene-FISH allows the integration of genetic linkage maps and quantitative chromosome maps. Visualization methods using FISH can reveal the spatial organization of the centromere, heterochromatin/euchromatin, and the terminal structures of rice chromosomes. Furthermore, EDF-FISH and the DNA combing technique can resolve a spatial distance of 1 kb between adjacent DNA sequences, and the detection of even a 300-bp target is now feasible. The copy numbers of various repetitive sequences and the sizes of various DNA molecules were quantitatively measured using the molecular combing technique. This review describes the significance of these advances in molecular cytology in rice and discusses future applications in plant studies using visualization techniques.

Application of the Chromosome Image Analyzing System (CHIAS) for Straightening Cation-treated Bent Chromosomes.

Divalent cations, mainly calcium and magnesium ions, are known to play a major role in the maintenance of chromosomes. The depletion of both ions using ethylenediaminetetraacetic acid (EDTA) results in a bent chromosome structure with extended arms and dispersed chromatin fibers. The importance of divalent cations for the maintenance of chromosome structure has been reported previously; nevertheless, previous studies were limited to qualitative data only. Straightening the bent image of the chromosome would provide quantitative data. Thus, this study aimed to evaluate the effects of cation depletion by the application of the Chromosome Image Analyzing System (CHIAS) to straighten bent chromosomes. Human HeLa chromosomes were treated with EDTA as a known chelating agent in order to investigate the importance of divalent cations on the maintenance of chromosome structure. Chromosomes were stained and directly observed with a fluorescence microscope. Images were then analyzed using CHIAS. The results revealed that EDTA-treated chromosomes showed longer arms than those without EDTA treatment, and most of them tended to bend-out. By straightening the image using CHIAS, the bent chromosomes were successfully straightened. The average lengths of the chromosomes treated with and without EDTA were 4.97 and 0.96 µm, respectively. These results signify the advantages of CHIAS for chromosome analysis and highlight the fundamental effects of cations on chromosome condensation.