Planta DOI 10.1007/s00425-014-2233-9

REVIEW

Immuno-cytogenetic manifestation of epigenetic chromatin modification marks in plants Santosh Kumar Sharma • Maki Yamamoto Yasuhiko Mukai

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Received: 1 September 2014 / Accepted: 16 December 2014 Ó Springer-Verlag Berlin Heidelberg 2014

Abstract Histone proteins and the nucleosomes along with DNA are the essential components of eukaryotic chromatin. Post-translational histone–DNA interactions and modifications eventually offer significant alteration in the chromatin environment and potentially influence diverse fundamental biological processes, some of which are known to be epigenetically inherited and constitute the ‘‘epigenetic code’’. Such chromatin modifications evidently uncover remarkable diversity and biological specificity associated with distinct patterns of covalent histone marks. The past few years have witnessed major breakthroughs in plant biology research by utilizing chromatin modificationspecific antibodies through molecular cytogenetic tools to ascertain hallmark signatures of chromatin domains on the chromosomes. Here, we survey current information on chromosomal distribution patterns of chromatin modifications with special emphasis on histone methylation, acetylation, phosphorylation, and centromere-specific histone 3 (CENH3) marks in plants using immuno-FISH as a basic tool. Major available information has been classified under typical and comparative cytogenetic detection of chromatin modifications in plants. Further, spatial distribution of chromatin environment that exists between different cell Electronic supplementary material The online version of this article (doi:10.1007/s00425-014-2233-9) contains supplementary material, which is available to authorized users. S. K. Sharma (&)  Y. Mukai Division of Natural Sciences, Laboratory of Plant Molecular Genetics, Osaka Kyoiku University, 4-698-1 Asahigaoka, Kashiwara Osaka 582-8582, Japan e-mail: [email protected]; [email protected] M. Yamamoto Department of Rehabilitation Sciences, Kansai University of Welfare Sciences, Kashiwara, Osaka 582-0026, Japan

types such as angiosperm/gymnosperm, monocot/dicot, diploid/polyploids, vegetative/generative cells, as well as different stages, i.e., mitosis versus meiosis has also been discussed in detail. Several challenges and future perspectives of molecular cytogenetics in the grooming field of plant chromatin dynamics have also been addressed. Keywords DNA methylation  Histone modification  CENH3  Immuno-FISH  Plants

Introduction Chromatin is the state in which DNA is packaged within the cell. The nucleosome along with highly conserved histone proteins is central component of the chromatin. Histones, as the structural core of the nucleosome, are subjected to multiple types of post-translational covalent modifications, such as acetylation, phosphorylation, methylation, ubiquitylation, ADP-ribosylation (Jenuwein and Allis 2001) and sumoylation (Shiio and Eisenman 2003). Such high-order chromatin structure has an immense importance in monitoring gene expression and upholding the genome integrity. Histone variants and their modifications may alter chromatin structure by influencing histone– DNA and histone–histone interactions. It is also well documented fact that post-translational modifications on different histone tails act as recognition sequences for binding of different chromatin-associated factors and represent additional epigenetic information on chromatin, therefore proposed as a ‘‘histone code’’ for regulating transcriptional activity of an embedded gene (Strahl and Allis 2000). The histone code is likely to be universal as many of the chromatin proteins described in other organisms have a homolog in plants. In view of such evidence,

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understanding the relation between DNA sequence, chromatin proteins and chromosome structure, especially with respect to the dynamics of rapidly evolving centromeric chromatin that apparently exhibits signatures of adaptive evolution emerged as a scorching research area among plant biologist worldwide. In particular, our knowledge of the functional implication of histone tail modifications has rapidly increased but still in its infant stage. The diversified chromosomal location of ‘‘remembered’’ epigenetic regulatory histone/DNA marks allows us to catalog the cytological consequences underlying genome evolution. The global pattern of chromatin modifications along with nuclear size and shape, relative content and distribution of heterochromatin/euchromatin, organization and structure of chromosomes with respect to position and orientation may provide new insights into chromatin structure vis-a`-vis genome organization of the particular plant species at chromosomal level. In the last decade, molecular cytogenetics has acquired a prominent share in this newly developing research field of studying chromatin dynamics through epigenetic signatures of histone/DNA modifications. Significant evolution in molecular cytogenetic techniques especially immuno-FISH allows rapid and precise detection of chromatin modification marks in the organism under investigation. In 1989, the first antiserum for site-specific acetylation of histone H4 was generated by immunization with synthetic peptides (Turner et al. 1989). Since then, several antibodies for histone site-specific modifications (mostly methylation, acetylation and phosphorylation) have been generated and widely used for in situ detection of DNA/ histone modification marks underlying chromatin status and their chromosomal distribution pattern in plants too. Briefly, immuno-FISH utilizes the isolation and fixation of plant materials (root tips/anthers/etc.) in 4 % paraformaldehyde in phosphate-buffered saline (PBS). Subsequently, enzymatic digestion (e.g., cellulose, pectinase and pectolyase, etc.) is carried out at 37 °C for several minutes (depends on plant material) followed by squash preparation of plant materials in PBS. The slides are then blocked for 30 min in 4 % (w/v) bovine serum albumin (BSA) plus 0.1 % Triton X-100 in PBS and incubated overnight with the primary antibody of desired histone modification in humid chamber at 4 °C. The slides are then incubated with affinity-purified fluorophoreconjugated polyclonal anti-rabbit/rat/mouse/goat secondary antibody against primary antisera for 1 h at 37 °C. After several washing steps in PBS, the preparations are counterstained with 40 ,6-diamidino-2-phenyl-indole (DAPI) along with anti-fade solution. An epifluorescence microscope is used for signal detection and image analysis. In plants, the distribution of eu/heterochromatin content is not uniform among chromosomes and can vary within and between chromosomes of a species. Mostly, 5-methyl cytosine (5-mCyt or 5-mC) is the principal modified base

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and DNA methylation mark, which account for cytosine residues in plants (Gruenbaum et al. 1981). In contrast, posttranslational modifications of histone N-terminal tails (Fig. 1) affect nucleosome positioning and compaction, and thus play pivotal role in chromatin remodeling and gene regulation (Kouzarides 2007). N-terminal lysine residues of histone H3 (K9, K14, K18 and K23) and H4 (K5, K8, K12, K16 and K20) are found to be acetylation/deacetylation targets, while histone H3 (K4, K9, K27 and K36) and H4 (K5, K8, K12 and K16) are predominantly targets for methylation in plant (Houben et al. 2007; Earley et al. 2007; Zhou 2009). Similarly, H3 serines (S10 and S28) and threonines (T3 and T11) are the known targets for phosphorylation and their chromosomal distribution can differ between groups of eukaryotes (Houben et al. 1999, 2007). Interestingly, Zhang et al. (2007) suggested some unique histone modifications in model plant system, i.e., Arabidopsis thaliana. The lysine residues of histone H2A (K144), and serine residues (S129, S141 and S145) were found to be acetylation and phosphorylation targets, respectively. On the other hand, histone H2B showed K6, K11, K27, K32 acetylation, S15 phosphorylation and K143 ubiquitination in plants (Zhang et al. 2007). Further, arginine methylation of histone H4 (H4R3) has also been reported recently (Yue et al. 2013). The major histone tail modifications commonly characterized in plants have been illustrated in Fig. 1 and important histone methylation marks that found to be associated with hetero/eu-chromatin in various plant species have been listed in supplementary Table S1. Further, centromere-specific histone H3 (CENH3) also known as one of the rapidly evolving most fundamental centromeric proteins is known to be involved in chromatin remodeling (Jiang et al. 2003; Nagaki et al. 2005). Post-translational modifications in CENH3 could show considerable variability between species and epigenetic signs of adaptive evolution (Houben and Schubert 2003). Several landmark immuno-cytogenetic studies have already been conducted by employing antibodies against such histone variants to illuminate their distribution pattern on plant chromosomes. The specific cytogenetic distribution pattern and behavior of histone marks are often epigenetic regulated events during evolution and raise important questions to be answered in the future. Through this review, an attempt has been made to collect, collate and update the major information existing on typical chromosomal distribution and behavior of the epigenetic regulatory chromatin signatures of post-translational DNA/ histone modifications in plants. Further, comparative distribution of histone marks targeting monocot versus dicot, diploid versus polyploid and/or hybrids, mitosis versus meiosis, vegetative versus generative nucleus in pollens and A versus B chromosomes has also been addressed to understand the underlying impact of the chromatin modification on plant genome organization and evolution.

Planta Fig. 1 A Diagrammatical representation of chromatin organization with histone tail modification including (a) typical nucleosome assembly showing high-order DNA compaction and folding (b) a histone octamer that consists of pair of each of the four core histone proteins (H2A, H2B, H3 and H4), N-terminus tail of each histone protein represents amino acid residues at specific positions that are subject to a number of posttranslational modifications such as methylation, acetylation, phosphorylation and ubiquitination (c) unmodified H3 and H4 histone proteins having a globular domain and N-terminus tail (d) Modified H3 and H4 histone proteins. B Diagrammatical representation of histone tail modification reported in plants. Relevant histone proteins that are known to interact or associate with distinct modification (methylation, acetylation, phosphorylation and ubiquitination) patterns are indicated at the respective place of occurrence with special references to the plants

Typical distribution pattern of chromatin environment in plants Several plant species have been evaluated molecular cytogenetically to characterize chromatin environments including both monocots and dicots. The survey of the literature revealed that much focus has been given to monocots over dicot plants. Among them, Vicia faba was first characterized for the distribution of H4 acetylated isoforms at chromosomal level. The acetylation at positions of lysine 5, 8, and 12 was correlated with the intensity of transcription (Houben et al. 1996). Further, Histone H3 was also found under-acetylated in some heterochromatic regions and subsequent differential acetylation patterns of

H4 and H3 were identified by Belyaev et al. (1998). Similarly, H3K9ac was also found to be associated with actively transcribed genes and influence numerous developmental and biological processes in higher plants (Ausı´n et al. 2004; Servet et al. 2008). The accumulation of H3K9me and the hypermethylation of DNA have been typically attributed to consecutive heterochromatin, whereas DNA hypomethylation and the presence of H3K4me were considered to be characteristic of euchromatin as evidently revealed in several studies on plants (Jenuwein and Allis 2001; Jasencakova et al. 2003; Jackson et al. 2004; Suzuki et al. 2010; Nagaki et al. 2004, 2005, 2009, 2012a, b). Additionally, immunostaining with antibodies that discriminate between mono-, di- and tri-

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methylation of specific lysines (K4, K9, K27 and K36 of H3 and K20 of H4) identified H3K4me1,2,3 and H3K36me1,2,3, together with H3K9me3, H3K27me3 and H4K20me2,3 as typical marks for euchromatin. However, the heterochromatic chromocenters of Arabidopsis were found to be enriched in H3K9me1,2, H3K27me1,2 and H4K20me1 (Soppe et al. 2002; Jackson et al. 2004; Houben et al. 2007). Shi and Dawe (2006) used immunofluorescence assay on maize meiotic pachytene chromosomes and suggested that H3K27me2 also marks classical heterochromatin and favors the H3K4me2 as euchromatic mark. However, in contrary, H3K9me2 was not associated with consecutive heterochromatic regions and behaved like H3K4me2 and H3K27me3 by marking euchromatin. Histone H4K20me2,3 was reported to be absent in maize. Further, Jin et al. (2008) confirmed the association of acetylated H3/H4 and H3K27me1 with eu- and heterochromatic segments of maize chromosomes, respectively. Immuno-fluorescent detection of 5-mC on super-stretched pachytene chromosomes was characteristically found to be associated with the centromeric and pericentromeric regions of maize chromosome (Koo and Jiang 2009). Suzuki et al. (2010) revealed the distribution pattern of H3K4me2, H3K9me2, H3K27me1 and H3K27me3 on onion chromosomes and confirmed that onion genomic DNA is highly methylated with the distribution of methylated CG dinucleotide on entire chromosomes. It was also opined that the distribution of histone methylation codes is closely related to those of large genome plant species. Recently, He et al. (2014) also advocated the conserved distribution pattern of identified histone modifications along the chromosomes of cultivated and wild maize using a three-dimensional (3D) epigenome karyotyping approach by combining immunostaining and 3D reconstruction with deconvolution techniques. On the other hand, phosphorylation of H3 serines (S10 and S28) and threonines (T3 and T11) were correlated with the position of the peri-centromere during mitosis and meiosis II in plants and their chromosomal distribution was found to be different between groups of eukaryotes (Houben et al. 1999, 2007). Similarly, phosphorylated histone H2AT133 (equivalent to human H2AT120) was immuno-cytogenetically characterized on maize chromosomes and showed affinity towards centromeric regions during mitosis (Dong and Han 2012). Recently, H2AT120ph has also been shown to be evolutionarily highly conserved across species with mono-/holocentric chromosomes and thus proposed as a universal marker for the cytological detection of centromeres of mono- and holo-kinetic plant species (Demidov et al. 2014). To date, there are no data on the chromosomal distribution of H3K14,18,23 methylation and the methylated terminal arginines of H3 and H4 along with H3 threonines 6, 22 in plants.

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Investigations on structure and function of plant CENH3s have been mostly reported in monocot plants; however, a few reports are also available for dicot plant species including Nicotiana tabacum (Nagaki et al. 2009, 2011), some Brassica species (Wang et al. 2011) and several members of the Leguminosae family including soybean, common bean, and peas (Tek et al. 2010, 2011; Neumann et al. 2012; Iwata et al. 2013) and cultivated and wild Daucus species (Dunemann et al. 2014). Monocots include Zea mays (Zhong et al. 2002), Oryza sativa (Nagaki et al. 2004; Hirsch et al. 2009), Saccharum officinarum (Nagaki and Murata 2005), Hordeum species (Sanei et al. 2011) and Allium species (Nagaki et al. 2012b). Several reports suggest that the centromeric regions of Arabidopsis chromosomes are composed of distinct heterochromatin. The centromeric heterochromatin forms the chromocenters in interphase nuclei and is highly enriched with H3K9me2. The DNA sequences in the centromeric regions were extensively methylated as revealed through cytological investigations (Soppe et al. 2002; Jasencakova et al. 2003; Probst et al. 2003). However, later Zhang et al. (2008) observed hypomethylation in 178-bp repeats associated with the CENH3-containing chromatin (CEN chromatin) compared with the same repeats located in the flanking pericentromeric regions. An immunofluorescence assay was performed using an antibody against 5-mC which observed bright and uniform signals associated with the chromocenters in most cells unlikely non-uniform and hollow-heart signals in some large nuclei. Further, it was confirmed that hollow centers were actually CENH3associated centromeric chromatin as reveled using an antiCENH3 antibody. Such results were also confirmed on super-stretched pachytene chromosomes of maize. A cumulative data analysis suggested that the DNA sequences in the CENH3-associated regions might be differentially methylated in comparison to the flanking pericentromeric regions. Nagaki and Murata (2005) characterized the CENH3associated DNA sequences in sugarcane. Subsequently, Nagaki et al. (2005), in an effort to determine the holocentric structure in plants, successfully developed a putative centromere-specific histone H3 antibody against Luzula nivea (LnCENH3). Immunostaining clearly revealed the diffuse centromere-like structure that appears in the linear shape at prophase to telophase. Furthermore, it was shown that the amount of LnCENH3 decreased significantly at interphase. The polar side positioning on each chromatid at metaphase to anaphase also confirmed that LnCENH3 represents one of the centromere-specific histone proteins in L. nivea. Further, same group (Nagaki et al. 2009) characterized the CENH3 in tobacco and revealed that a centromeric DNA sequence is located in the vicinity of NtCENH3 in tobacco. To identify and characterize the

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CENH3 variants in Allium, Nagaki et al. (2012b) surveyed CENH3-coding cDNAs in four Allium species and a polyclonal antibody against CENH3 of welsh onion recognized all orthologs. Immunostaining of Allium chromosomes using anti-Afi-CENH3 and centromeric localization of Chromatin Immuno-Precipitation (ChIP) clones is shown in Fig. 2. Additionally, three-dimensional (3D) observation of mitotic cell division using immuno-histochemical technique demonstrated 3D dynamics of the cells and position of cell-cycle marker proteins (CENH3 and atubulin). Such analysis established the overall in situ location and direction of the dividing cells thereby improving the understanding of plant cellular organization and cell division in plants. Recently, Tek et al. (2014) characterized the Lotus japonicus homolog of the CenH3 gene (LjCenH3) encoding a 159-amino acid protein. The LjCenH3 was tagged with a green fluorescent protein and transferred into L. japonicus cells using an agrobacteriumbased transformation system. The centromeric position of LjCENH3 was confirmed on L. japonicus metaphase chromosomes by an immunofluorescence assay. It was opined that critical centromere landmark such as CENH3 could provide a better understanding of centromere structure in the various agriculturally important plant species. Further, in several cases, anti-CENH3 antibodies have recognized CENH3s of more or less closely related species. The transferability of antibodies synthesized against rice CENH3 (Nagaki et al. 2004) has been demonstrated in other Oryza species (Lee et al. 2005) including other members of Poaceae family such as barley (Houben et al. 2007), wheat (Liu et al. 2008), and rye (Houben et al. 2011). Similarly, CENH3 antibody transferability was also observed between different Brassica species (Nagaki et al. 2009) and several Allium species (Nagaki et al. 2012b) suggesting conserved nature of CENH3 at family level.

However, still the cross-transferability of antibodies among genera/families has not been reported yet. Ravi and Chan (2010, 2013) demonstrated the centromere-mediated genome elimination processes for the generation of haploid and double-haploid plants using Arabidopsis as a modal plant system. The study revealed that by manipulating the CENH3 in one of the parents (i.e., haploid inducer), formation of haploids could be induced (Ravi and Chan 2010). Such strategy of uniparental genome elimination has been advised to examine in other plants also to large-scale haploid production targeting crop breeding programs (Ravi and Chan 2010, 2013). Recently, Fu et al. (2013) demonstrated the de novo formation of centromere on a chromosome fragment in maize. This small chromosome has no detectable canonical centromeric sequences, but contained a site with protein features of functional centromeres such as CENH3, as detected by anti-CENH3 antibody and a foundational kinetochore protein, i.e., CENP-C. Such innovative studies accelerate the initiation, formation and subsequent developmental pathways of centromere sites on the chromatin fragment in plants. Beside characteristic distribution, several studies have also been carried out with special focus on comparative assessment of cytogenetic localization and distribution of chromatin modification marks. Most of such comparative assessments have been briefly described to understand and broaden our insight into large-scale genome organization in plants. Angiosperm versus gymnosperm: chromosomal distribution of critical histone marks is more or less conserved, however, minor deviations can occur Fuchs et al. (2008) analyzed the chromosomal distribution of seven histone methylation marks (H3K4me2, H3K9me1,2,3

Fig. 2 a Immunostaining of chromosomes of Allium species using anti-AfiCENH3 antibody: DAPI stained chromosomes (blue) and visualization of immuno-signals of antiAfiCENH3 antibody (red); b centromeric localization of DNA sequences isolated from A. fistulosum using chromatin immuno-precipitation (ChIP) clones: DAPI stained chromosomes of A. fistulosum (blue), rDNA signals (green) and FISH signals of ChIP clones (red)

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and H3K27me1,2,3) in the gymnosperm species Pinus sylvestris and Picea abies. The chromosomal distribution patterns of these histone methylation marks were compared to that of angiosperms. Histone H3K4me2 was found to be associated with euchromatin in both gymnosperms and angiosperms. However, H3K9me1 mark commonly classified as heterochromatin-specific in angiosperms was found to be restricted to the euchromatin in gymnosperms (P. sylvestris and P. abies). Most of the other histone methylation marks behaved similarly as reported in the angiosperm, for example, H3K9me2,3 and H3K27me1 were distributed equally along the chromosomes and the H3K9me3 and H3K27me2,3 were found to be associated with specific type of heterochromatin. The histone methylation marks more or less conserved in distribution pattern, however, revealed species-specificity, e.g., H3K9me3 showed enriched heterochromatin pattern in P. sylvestris compared to P. abies. It was concluded that although the histone methylation marks are conserved between eukaryotes, the distribution and functional specificity might have diverged and/or attributed during evolution. Therefore, several species of the same genus and/ or taxa having closed phylogenetic relationships may divulge in the chromosomal distribution of individual heterochromatin-associated histone methylation marks. Monocot versus dicot: variety of chromatin environment exists Vyskot et al. (1998), as one of the pioneering studies of comparative chromosomal distribution of H4K5ac, choose the monocot Allium cepa and the dicot Nicotiana tabacum and Silene spp. The antiserum displayed variable spectra on metaphase chromosomes. It was opined that histone acetylation and replication timing patterns could be useful landmarks for regions containing potentially active gene regions to map plant chromosomes. Houben et al. (2003) carried out detailed assessment of distribution pattern of histone modifications in 24 plant species including 10 monocots and 13 dicot plants. The distribution pattern of H3K4me and H3K9me indicated the typical labeling targeted at euchromatin and heterochromatin, respectively, without influence of mono or dicot status of plant taxa. This study also suggested that eu-/heterochromatin depends on genome size irrespective of mono-/di-cotyledon status of the plant. Further, H3K27me2,3 showed a species-specific chromosomal distribution when compared between monocot and dicot plants. H3K27me2 was typical for heterochromatin in Arabidopsis but tagged euchromatin in barley, whereas H3K27me3 is euchromatin-specific in Arabidopsis and barley but clusters at certain heterochromatic positions in V. faba (Fuchs et al. 2006). Recently, Demidov et al. (2014) analyzed chromosomal location of

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H2AT120ph in 20 different monocot and eudicot species and found conserved distribution across all the species having either mono- or holo-centric chromosomes and proposed H2AT120ph as a universal centromere marker system for the plant species. Diploid versus polyploids and hybrids: Polyploidy and hybridization do not affect the chromatin configuration A very little is known about histone modification patterns in polyploids, however, it is reported that allopolyploid formation leads to heritable re-patterned cytosine methylation as evidently revealed in several plant species. Such alterations in DNA methylation patterns were found to be conserved between particular polyploid individuals and even between synthetic and natural polyploids (Shaked et al. 2001; Liu and Wendel 2003). With the special concern to the centromere-specific histone, Talbert et al. (2002) demonstrated the labeling of antibodies specific against Arabidopsis thaliana CENH3 on centromere of A. suecica, a natural allotetraploid of A. thaliana and A. arenosa as well as their synthetic allotetraploid too which indicate the AtCENH3’s recognition of centromeric pattern of both parental species. Further, the lineage specificity and adaptive evolution of rice CENH3 with special reference to centromere repeats and polyploidy was reported by Hirsch et al. (2009). Further, Wang et al. (2011) elucidate the behavior of centromeric chromatin in the evolution of polyploidy by isolating the CENH3 (BrCENH3) genes in three diploid Brassica species and analyzed the sequence variation and expression level of these BrCENH3 genes in the allopolyploid hybrids Brassica juncea (AABB), B. napus (AACC), and B. carinata (BBCC). In addition, chromatin immuno-precipitation and immuno-labeling experiments with antiBrCENH3 antibodies indicated the conserve nature of BrCENH3 proteins. Such observations recommend that the allopolyploidization events did not alter the expression or evolutionary patterns of the CenH3 genes in plant taxa. In addition, the role of the CENH3 in the process of chromosome elimination was drawn using molecular cytogenetic characterization of interphase nucleus, anaphase chromosomes of an unstable and stable Hordeum vulgare 9 H. bulbosum hybrid embryo using an anti-grass CENH3 and anti–a-tubulin (Sanei et al. 2011). The observation identified the two functional CENH3s in both diploid barley species and their incorporation into centromeres of alien chromosomes was carried out. The study suggested that if multiple CENH3s coexist in species combinations, all parental CENH3 variants might not be associated with centromeres of hybrid and ultimately can act as a barrier to species hybridization.

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To compare the differences between heterochromatin and euchromatin epigenetic marks, Braszewska-Zalewska et al. (2010) carried out an interesting comparative cytogenetic analysis of DNA and histone H3 methylation patterns in the interphase nuclei of diploid and allotetraploid Brassica species. The observation revealed the differences in the patterns of DNA and histone H3 methylation between them. The most prominent differences were attributed to diploid brassicas. DNA methylation was present exclusively in the heterochromatin only in B. rapa. In B. oleracea and B. napus, this modification was detected in both euchromatin and heterochromatin. A similar pattern was observed for H3K9me2. The H3K4me2 was found to be a typical marker of euchromatin in Brassica species as also suggested for other plant species. It was authentically concluded that the diploid species differ in patterns of analyzed epigenetic modifications and the allotetraploid B. napus has combined patterns from both diploids. Differences in patterns of DNA and histone H3 methylation between Brassica species were correlated with the genome structure and heterochromatin localization rather than ploidy level. Recently, a unique diploid versus autotetraploid paired sets of eight diverse clones differing in the qualitative composition of their native secondary metabolites belonging to six species of Cymbopogon were undertaken to understand the role of native secondary metabolites with reference to body size and ploidy change. In situ immunodetection of 5-mC sites revealed DNA methylation enhancement in autopolyploids and confirmed that whole genome duplication leads to autopolyploidy and brings about an increase in cell size, concentration of secondary metabolites and enhanced cytosine methylation (Lavania et al. 2012). Mitosis versus meiosis: histone phosphorylation is the key player Phosphorylation is one of the well-known post-translational histone modifications and correlated to chromosome condensation. Histone H3 is highly phosphorylated when the cell enters mitosis. The histone H3S10 is well characterized to be tightly associated with mitosis in many organisms including plants (Houben et al. 2005; Fuchs et al. 2006). This phosphorylation is believed to be a crucial step in the high orders of chromatin condensation and compaction, which are essential for subsequent chromosome congression and segregation during mitosis and meiosis. In plants, the distribution of H3S10ph and H3S28ph is restricted to pericentromeric regions during mitosis and meiosis II (Houben et al. 1999). It was also observed that during the first meiotic division both residues are phosphorylated along the entire length of the chromosomes but H3 phosphorylation became absent during

meiosis II (Houben et al. 2007; Xu et al. 2009). These observations led to the hypothesis that, in plants, pericentromeric H3 phosphorylation at both serine positions is required for cohesion of sister chromatids during metaphase I, and for cohesion of sister pericentromeres during mitosis and metaphase II (Houben et al. 2007). Such information was significantly updated by Caperta et al. (2008) who studied the distribution patterns of phosphorylated histone H3T3 and H3T32 in mitosis and meiosis of both higher genome size plant species (Secale cereale, V. faba and Hordeum vulgare) and small genome species Arabidopsis thaliana. The study reveled that both histone modifications (H3T3/T32) can be traced out from diakinesis to anaphase I (first meiotic division) as well as metaphase II to anaphase II (second meiotic division) in large genome species, whereas small genome species (Arabidopsis) showed dephosphorylation at anaphase I and II. Both histone modifications were distributed along the entire length of chromosomes during mitotic metaphase and metaphase I; however, H3T3 was restricted to the pericentromeric domain during second meiotic division, while H3T32 showed presence all along the chromosome arms of all species analyzed. In addition, Dong and Han (2012) demonstrated the localization of H2AT133 on maize chromosome both at mitotic and meiotic divisions. This study established the specificity of H2AT133ph towards centromeric regions of the chromosomes. During mitosis, H2AT133ph was found to be strong in metaphase decreased during later anaphase and telophase. Further, maize dicentric chromosomes revealed that the inactive centromeres have lost H2AT133ph. During meiosis, H2A phosphorylation was strong in the early pachytene stage and showed increase intensities up to metaphase I. Beside phosphorylation, Oliver et al. (2013) analyzed the comparative chromosomal distribution of selected histone variants associated with transcriptional regulation and differentiation of eu/heterochromatin throughout all phases of both meiosis and mitosis in A. thaliana, Secale cereale and Aegilops sp. The immunofluorescence signals of H3 acetylation and methylation were held constant throughout mitosis and meiosis in all cases with a little divergence in case of acetylation, revealing the existence of speciesspecific chromatin-based regulatory mechanisms. Vegetative versus generative nuclei of pollen: chromatin organization is not universal Several antibodies recognizing post-translational histone modifications were assayed to identify epigenetic differences between generative and vegetative nuclei of the pollen grains in plant species. Janousek et al. (2000) reported high level of H4K5&8 in the generative nucleus of the mature pollen grain of Lilium longiflorum. Similarly,

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histone H3 acetylation and H3K4 methylation were found to be greater in the generative nucleus than that in the vegetative nucleus of lily. However, the euchromatin-specific mark H3K4me2 was found to be weakly represented in generative cell chromatin. An increase of H3K27me3 was found to spread with pollen maturation across the euchromatic regions of vegetative nuclei of lily, which suggests that H3K27me3 controls gene expression in the vegetative cell (Okada et al. 2006; Sano and Tanaka 2010). Such reports suggest that male gametic cells of Lilium have unique chromatin state and methylation of histone H3 variants might have involved in gene regulation of male gametic cells. In contrast to those observation, Ribeiro et al. (2009) reported the consistency in high level of 5-mC and H3K9me2 in generative cells whereas H3K9me3 and H4 K acetylation in vegetative cells of Quercus suber pollen. Comparative distribution of 5-mC in vegetative and generative nuclei from Q. suber and Lilium sp. pollen grains also confirmed the hypermethylation in the vegetative than in the generative nucleus. Similarly, Houben et al. (2011) reported the enhanced levels of histone H3K4/ K9me2 and H3K9ac in condensed chromatin of generative nuclei during first and second pollen mitosis in rye. Interestingly, in contrast to the previous reports of Okada et al. (2006) and Sano and Tanaka (2010), H3K27me3 was not found to be involved in transcriptional downregulation of genes located in generative nuclei. The constant level of DNA methylation in both types of pollen nuclei led the hypothesis that the chromatin organization in mature nuclei of pollen grains is not universal across angiosperms and subjected to differential expression. Such hypothesis received support from the studies by Pandey et al. (2013) who identified the reduced gene activity in the generative nucleus and increased expression in the vegetative nucleus during pollen development in barley. A versus B chromosomes: differential signal intensities of chromatin modification B chromosomes are dispensable components of the genomes of numerous species. Houben et al. (1997) observed the differences in labeling intensities in A and B chromosomes of Brachycome dichromosomatica with antibodies specific for different acetylated forms (lysine 5, 8, 12 and 16) of histone H4. The H4K5ac and H4K8ac were found to be labeled brightly in the A chromosomes, whereas inactive B chromosomes were faintly labeled. Carchilan et al. (2007) assayed H3K4/K9/K27me1,2,3 to A and B chromosomes of rye (Secale cereale) and observed that H3K9me1 and 3 marked heterochromatin as reported for H3K9me2 with uniform distribution throughout chromosome arms. H3K27me1 shows a species-specific chromosomal distribution and the euchromatic regions of rye As

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and Bs are uniformly H3K27me1 labeled, while the terminal heterochromatic chromosomal regions were found to be enriched in H3K27me2,3. The H4K20me1,2,3 showed very weak signal; however, 5-methylcytosine DNA showed a punctuated and uniform pattern along both the As and Bs, without any particular sites of accumulation. Koo et al. (2011) observed distinct pattern of DNA methylation that was associated with active and inactive centromeres of the maize B chromosome using immuno-FISH approach. Such studies targeting A and B chromosomes revealed distinct DNA methylation and histone modification pattern which needs to be further investigated in other plant species having B chromosomes to understand the cytogenetic perspective of epigenetic evolution, organization and function of supernumerary/accessory chromosomes in plants.

Challenges and future perspectives One of the major challenges includes the increment of the resolution power of in situ hybridization and immunostaining techniques to detect shorter nucleotide stretches or single antigen molecules reliably on metaphase chromosomes, extended chromatin fibers and/or in interphase nuclei. Further, improvement of efficient and effective fluorescent chromatin tags is also needed. Such technical improvements will be helpful to resolve the chromosome structure and organization during cell divisions. It may also shed light on chromatin dynamics during mitosis and meiosis with reference to replication, transcription, repair, or recombination processes. Although almost 25 years has passed since the first antibody against a histone sites-specific modification was developed, the availability and affordability of such antibodies is still a limiting factor in this area of in situ chromosomal detection of plant chromatin modification. Several commercially produced antibodies are available but expensive and their quality varies from batch to batch. Thus, an inexpensive and reliable source of high affinity antibodies against specific histone modifications is important for this area of research. Further, only a few histone variants and centromeric histone proteins have been cytogenetically characterized in a limited number of plant species/families that needs to be extended immensely. Very importantly, phospho-specific antibodies are still unsatisfactory to discriminate the phosphorylation status of H3 variants. Future work should also be focused on exploring histone H3 phosphorylation and other histone marks that are still unidentified in plants to generate a huge dataset to understand the epigenetic language of chromatin modification in plant evolution. Several epigenetic regulatory histone/DNA modifications play a major role in plant

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phenotype development with reference to transcription, development plasticity and interactions with the changing environment. Therefore, it would be an important aspect to know about the relationship between the distribution of histone variants on the chromosomes and its ability to identify trait loci showing recombination events in segregating population. Moreover, the distribution and dynamics of these modifications seem to be species-specific and even differ between mitosis and meiosis in the same species. Further research should be focused on analysis of distribution of DNA/histone modification marks, its unique localization and special features if any, targeting the plant species with various chromosome numbers, species complex, different ploidy levels as well as taxonomic complications. Additionally, epigenetic variations commonly generate at a much higher rate than equivalent genetic variation, especially in rapidly changing environmental conditions. In this context, Granot et al. (2009) demonstrated the association of histone modifications with drought tolerance in the desert plant Zygophyllum dumosum. As per this study, the nuclear size and histone modification marks (e.g., H3K4me2, associated with active gene expression or euchromatin) were found to be higher during the wet season (active plant growth) but gradually reduced with progression to the dry season (inactive plant growth). Therefore, plant species, which are cosmopolitan in distribution along with diverse climate conditions and complex genome structure, should be analyzed at chromosome and genomic/epigenomic levels to understand the role of DNA/histone modifications with respect to speciation and evolution. The family Orchidaceae may serve as a model plant family to take such initiative, as orchids are found in almost all types of habitats in climates ranging from arctic to tropical, having various basic chromosome number and ploidy. Further, active role of chromatin modification in flowering time control has also been investigated thoroughly (He and Amasino 2005). Histone modifications such as H3K4me3 and H3K9/27me2 have been identified to associate with active FLOWERING LOCUS C (FLC) expression and repression, respectively. Such ‘regulation switch’ also influences the state of FLC chromatin and should also be taken into consideration to study chromatin dynamics with special reference to regulation of flowering time and longevity. Several highthroughput approaches such as mass spectrometry, next generation sequencing (NGS) technologies can also be combined to the immunological assays to decipher the genome-wide chromatin dynamics in plants. Beside this, functional analysis of other core histones including linker histones will definitely improve our knowledge on the complex interplay of post-translational histone modifications in plants.

Acknowledgments We thank Japan Society for the Promotion of Science (JSPS), Japan for providing postdoctoral fellowship and research grant (SKS, no. P13399/2013). Sincere thanks are also due to Dr. Go Suzuki and all members of Plant Molecular Genetics Laboratory, Osaka Kyoiku University, Osaka, Japan for their constant encouragement and help. We apologize to those authors whose works we could not cite because of space limitations. Conflict of Interest

Authors declare no conflict of interest.

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