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Single-stranded DNA (ssDNA) is essential for various DNA-templated processes in both eukaryotes and prokaryotes. However, comprehensive characterizations of ssDNA still lag in plants compared to nonplant systems. Here, we conducted in situ S1-sequencing, with starting gDNA ranging from 5 µg to 250 ng, followed by comprehensive characterizations of ssDNA in rice (Oryza sativa L.). We found that ssDNA loci were substantially associated with a subset of non-B DNA structures and functional genomic loci. Subtypes of ssDNA loci had distinct epigenetic features. Importantly, ssDNA may act alone or partly coordinate with non-B DNA structures, functional genomic loci, or epigenetic marks to actively or repressively modulate gene transcription, which is genomic region dependent and associated with the distinct accumulation of RNA Pol II. Moreover, distinct types of ssDNA had differential impacts on the activities and evolution of transposable elements (TEs) (especially common or conserved TEs) in the rice genome. Our study showcases an antibody-independent technique for characterizing non-B DNA structures or functional genomic loci in plants. It lays the groundwork and fills a crucial gap for further exploration of ssDNA, non-B DNA structures, or functional genomic loci, thereby advancing our understanding of their biology in plants., Competing Interests: Conflict of interest statement. None declared., (© The Author(s) 2024. Published by Oxford University Press on behalf of American Society of Plant Biologists. All rights reserved. For commercial re-use, please contact reprints@oup.com for reprints and translation rights for reprints. All other permissions can be obtained through our RightsLink service via the Permissions link on the article page on our site—for further information please contact journals.permissions@oup.com.)

I-motifs (iMs) are four-stranded non-B DNA structures containing C-rich DNA sequences. The formation of iMs is sensitive to pH conditions and DNA methylation, although the extent of which is still unknown in both humans and plants. To investigate this, we here conducted iMab antibody-based immunoprecipitation and sequencing (iM-IP-seq) along with bisulfite sequencing using CK (original genomic DNA without methylation-related treatments) and hypermethylated or demethylated DNA at both pH 5.5 and 7.0 in rice, establishing a link between pH, DNA methylation and iM formation on a genome-wide scale. We found that iMs folded at pH 7.0 displayed higher methylation levels than those formed at pH 5.5. DNA demethylation and hypermethylation differently influenced iM formation at pH 7.0 and 5.5. Importantly, CG hypo-DMRs (differentially methylated regions) and CHH (H = A, C and T) hyper-DMRs alone or coordinated with CG/CHG hyper-DMRs may play determinant roles in the regulation of pH dependent iM formation. Thus, our study shows that the nature of DNA sequences alone or combined with their methylation status plays critical roles in determining pH-dependent formation of iMs. It therefore deepens the understanding of the pH and methylation dependent modulation of iM formation, which has important biological implications and practical applications., (© The Author(s) 2024. Published by Oxford University Press on behalf of Nucleic Acids Research.)

A G-quadruplex (G4) is a typical non-B DNA structure and involved in various DNA-templated events in eukaryotic genomes. PEG and PDS chemicals have been widely applied for promoting the folding of in vivo or in vitro G4s. However, how PEG and PDS preferentially affect a subset of G4 formation genome-wide is still largely unknown. We here conducted a BG4-based IP-seq in vitro under K + +PEG or K + +PDS conditions in the rice genome. We found that PEG-favored IP-G4s + have distinct sequence features, distinct genomic distributions and distinct associations with TEGs, non-TEGs and subtypes of TEs compared to PDS-favored ones. Strikingly, PEG-specific IP-G4s + are associated with euchromatin with less enrichment levels of DNA methylation but with more enriched active histone marks, while PDS-specific IP-G4s + are associated with heterochromatin with higher enrichment levels of DNA methylation and repressive marks. Moreover, we found that genes with PEG-specific IP-G4s + are more expressed than those with PDS-specific IP-G4s + , suggesting that PEG/PDS-specific IP-G4s + alone or coordinating with epigenetic marks are involved in the regulation of the differential expression of related genes, therefore functioning in distinct biological processes. Thus, our study provides new insights into differential impacts of PEG and PDS on G4 formation, thereby advancing our understanding of G4 biology.

COLD is a major naturally occurring stress that usually causes complex symptoms and severe yield loss in crops. R-loops function in various cellular processes, including development and stress responses, in plants. However, how R-loops function in COLD responses is largely unknown in COLD susceptible crops like rice (Oryza sativa L.). We conducted DRIP-Seq along with other omics data (RNA-Seq, DNase-Seq and ChIP-Seq) in rice with or without COLD treatment. COLD treatment caused R-loop reprogramming across the genome. COLD-biased R-loops had higher GC content and novel motifs for the binding of distinct transcription factors (TFs). Moreover, R-loops can directly/indirectly modulate the transcription of a subset of COLD-responsive genes, which can be mediated by R-loop overlapping TF-centered or cis-regulatory element-related regulatory networks and lncRNAs, accounting for c. 60% of COLD-induced expression of differential genes in rice, which is different from the findings in Arabidopsis. We validated two R-loop loci with contrasting (negative/positive) roles in the regulation of two individual COLD-responsive gene expression, as potential targets for enhanced COLD resistance. Our study provides detailed evidence showing functions of R-loop reprogramming during COLD responses and provides some potential R-loop loci for genetic and epigenetic manipulation toward breeding of rice varieties with enhanced COLD tolerance., (© 2023 The Authors New Phytologist © 2023 New Phytologist Foundation.)

Cis-regulatory elements (CREs) fine-tune gene transcription in eukaryotes. CREs with sequence variations play vital roles in driving plant or crop domestication. However, how global sequence and structural variations (SVs) are responsible for multilevel changes between indica and japonica rice (Oryza sativa) is still not fully elucidated. To address this, we conducted multiomic studies using MNase hypersensitivity sequencing (MH-seq) in combination with RNA sequencing (RNA-seq), chromatin immunoprecipitation sequencing (ChIP-seq), and bisulfite sequencing (BS-seq) between the japonica rice variety Nipponbare (NIP) and indica rice variety 93-11. We found that differential MNase hypersensitive sites (MHSs) exhibited some distinct intrinsic genomic sequence features between NIP and 93-11. Notably, through MHS-genome-wide association studies (GWAS) integration, we found that key sequence variations may be associated with differences of agronomic traits between NIP and 93-11, which is partly achieved by MHSs harboring CREs. In addition, SV-derived differential MHSs caused by transposable element (TE) insertion, especially by noncommon TEs among rice varieties, were associated with genes with distinct functions, indicating that TE-driven gene neo- or subfunctionalization is mediated by changes of chromatin openness. This study thus provides insights into how sequence and genomic SVs control agronomic traits of NIP and 93-11; it also provides genome-editing targets for molecular breeding aiming at improving favorable agronomic properties., Competing Interests: Conflict of interest statement. The authors declare no competing intersects., (© American Society of Plant Biologists 2023. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.)

In hybrid plants, heterosis often produces large, vigorous plants with high yields; however, hybrid seeds are generated by costly and laborious crosses of inbred parents. Apomixis, in which a plant produces a clone of itself via asexual reproduction through seeds, may produce another revolution in plant biology. Recently, synthetic apomixis enabled clonal reproduction of F 1 hybrids through seeds in rice (Oryza sativa), but the inheritance of the synthetic apomixis trait and superior heterotic phenotypes across generations remained unclear. Here, we propagated clonal plants to the T 4 generation and investigated their genetic and molecular stability at each generation. By analyzing agronomic traits, as well as the genome, methylome, transcriptome, and allele-specific transcriptome, we showed that the descendant clonal plants remained stable. Unexpectedly, in addition to normal clonal seeds, the plants also produced a few aneuploids that had eliminated large genomic segments in each generation. Despite the identification of rare aneuploids, the observation that the synthetic apomixis trait is stably transmitted through multiple generations helps confirm the feasibility of using apomixis in the future., (Copyright © 2022 The Author(s). Published by Elsevier Inc. All rights reserved.)

Plants manage the high cost of immunity activation by suppressing the expression of defense genes during normal growth and rapidly switching them on upon pathogen invasion. TGAs are key transcription factors controlling the expression of defense genes. However, how TGAs function, especially in monocot plants like rice with continuously high levels of endogenous salicylic acid (SA) remains elusive. In this study, we characterized the role of OsTGA5 as a negative regulator of rice resistance against blast fungus by transcriptionally repressing the expression of various defense-related genes. Moreover, OsTGA5 repressed PTI responses and the accumulation of endogenous SA. Importantly, we showed that the nucleus-localized casein kinase II (CK2) complex interacts with and phosphorylates OsTGA5 on Ser-32, which reduces the affinity of OsTGA5 for the JIOsPR10 promoter, thereby alleviating the repression of JIOsPR10 transcription and increasing rice resistance. Furthermore, the in vivo phosphorylation of OsTGA5 Ser-32 was enhanced by blast fungus infection. The CK2 α subunit, depending on its kinase activity, positively regulated rice defense against blast fungus. Taken together, our results provide a mechanism for the role of OsTGA5 in negatively regulating the transcription of defense-related genes in rice and the repressive switch imposed by nuclear CK2-mediated phosphorylation during blast fungus invasion., (© The Author(s) 2022. Published by Oxford University Press on behalf of American Society of Plant Biologists.)

Growing evidence indicates that transposons or transposable elements (TEs)-derived accessible chromatin regions (ACRs) play essential roles in multiple biological processes by interacting with trans -acting factors. However, the function of TE-derived ACRs in the regulation of gene expression in the rice genome has not been well characterized. In this study, we examined the chromatin dynamics in six types of rice tissues and found that ~8% of ACRs were derived from TEs and exhibited distinct levels of accessibility and conservation as compared to those without TEs. TEs exhibited a TE subtype-dependent impact on ACR formation, which can be mediated by changes in the underlying DNA methylation levels. Moreover, we found that tissue-specific TE-derived ACRs might function in the tissue development through the modulation of nearby gene expression. Interestingly, many genes in domestication sweeps were found to overlap with TE-derived ACRs, suggesting their potential functions in the rice domestication. In addition, we found that the expression divergence of 1070 duplicate gene pairs were associated with TE-derived ACRs and had distinct distributions of TEs and ACRs around the transcription start sites (TSSs), which may experience different selection pressures. Thus, our study provides some insights into the biological implications of TE-derived ACRs in the rice genome. Our results imply that these ACRs are likely involved in the regulation of tissue development, rice domestication and functional divergence of duplicated genes.

DNA G-quadruplexes (G4s) are non-canonical four-stranded DNA structures involved in various biological processes in eukaryotes. Molecularly crowded solutions and monovalent cations have been reported to stabilize in vitro and in vivo G4 formation. However, how K + and Na + affect G4 formation genome-wide is still unclear in plants. Here, we conducted BG4-DNA-IP-seq, DNA immunoprecipitation with anti-BG4 antibody coupled with sequencing, under K + and Na + + PEG conditions in vitro. We found that K + -specific IP-G4s had a longer peak size, more GC and PQS content, and distinct AT and GC skews compared to Na + -specific IP-G4s. Moreover, K + - and Na + -specific IP-G4s exhibited differential subgenomic enrichment and distinct putative functional motifs for the binding of certain trans-factors. More importantly, we found that K + -specific IP-G4s were more associated with active marks, such as active histone marks, and low DNA methylation levels, as compared to Na + -specific IP-G4s; thus, K + -specific IP-G4s in combination with active chromatin features facilitate the expression of overlapping genes. In addition, K + - and Na + -specific IP-G4 overlapping genes exhibited differential GO (gene ontology) terms, suggesting they may have distinct biological relevance in rice. Thus, our study, for the first time, explores the effects of K + and Na + on global G4 formation in vitro, thereby providing valuable resources for functional G4 studies in rice. It will provide certain G4 loci for the biotechnological engineering of rice in the future.

Transposons significantly contribute to genome fractions in many plants. Although numerous transposon-related mutations have been identified, the evidence regarding transposon-derived genes regulating crop yield and other agronomic traits is very limited. In this study, we characterized a rice Harbinger transposon-derived gene called PANICLE NUMBER AND GRAIN SIZE (PANDA), which epigenetically coordinates panicle number and grain size. Mutation of PANDA caused reduced panicle number but increased grain size in rice, while transgenic plants overexpressing this gene showed the opposite phenotypic change. The PANDA-encoding protein can bind to the core polycomb repressive complex 2 (PRC2) components OsMSI1 and OsFIE2, and regulates the deposition of H3K27me3 in the target genes, thereby epigenetically repressing their expression. Among the target genes, both OsMADS55 and OsEMF1 were negative regulators of panicle number but positive regulators of grain size, partly explaining the involvement of PANDA in balancing panicle number and grain size. Moreover, moderate overexpression of PANDA driven by its own promoter in the indica rice cultivar can increase grain yield. Thus, our findings present a novel insight into the epigenetic control of rice yield traits by a Harbinger transposon-derived gene and provide its potential application for rice yield improvement., (© 2022 The Authors. Plant Biotechnology Journal published by Society for Experimental Biology and The Association of Applied Biologists and John Wiley & Sons Ltd.)

I-motifs (iMs) are non-canonical DNA secondary structures that fold from cytosine (C)-rich genomic DNA regions termed putative i-motif forming sequences (PiMFSs). The structure of iMs is stabilized by hemiprotonated C-C base pairs, and their functions are now suspected in key cellular processes in human cells such as genome stability and regulation of gene transcription. In plants, their biological relevance is still largely unknown. Here, we characterized PiMFSs with high potential for i-motif formation in the rice genome by developing and applying a protocol hinging on an iMab antibody-based immunoprecipitation (IP) coupled with high-throughput sequencing (seq), consequently termed iM-IP-seq. We found that PiMFSs had intrinsic subgenomic distributions, cis-regulatory functions and an intricate relationship with DNA methylation. We indeed found that the coordination of PiMFSs with DNA methylation may affect dynamics of transposable elements (TEs) among different cultivated Oryza subpopulations or during evolution of wild rice species. Collectively, our study provides first and unique insights into the biology of iMs in plants, with potential applications in plant biotechnology for improving important agronomic rice traits., (© The Author(s) 2022. Published by Oxford University Press on behalf of Nucleic Acids Research.)

A DNA G-quadruplex (G4) is a non-canonical four-stranded nucleic acid structure involved in many biological processes in mammals. The current knowledge on plant DNA G4s, however, is limited; whether and how DNA G4s impact gene expression in plants is still largely unknown. Here, we applied a protocol referred to as BG4-DNA-IP-seq followed by a comprehensive characterization of DNA G4s in rice (Oryza sativa L.); we next integrated dG4s (experimentally detectable G4s) with existing omics data and found that dG4s exhibited differential DNA methylation between transposable element (TE) and non-TE genes. dG4 regions displayed genic-dependent enrichment of epigenomic signatures; finally, we showed that these sites displayed a positive association with expression of DNA G4-containing genes when located at promoters, and a negative association when located in the gene body, suggesting localization-dependent promotional/repressive roles of DNA G4s in regulating gene transcription. This study reveals interrelations between DNA G4s and epigenomic signatures, as well as implicates DNA G4s in modulating gene transcription in rice. Our study provides valuable resources for the functional characterization or bioengineering of some of key DNA G4s in rice., (© American Society of Plant Biologists 2021. All rights reserved. For permissions, please email: journals.permissions@oup.com.)

Although lagging behind studies in humans and other mammals, studies of R-loops in plants have recently entered an exciting stage in which the roles of R-loops in gene expression, genome stability, epigenomic signatures, and plant development and stress responses are being elucidated. Here, we review the strengths and weaknesses of existing methodologies, which were largely developed for R-loop studies in mammals, and then discuss the potential challenges of applying these methodologies to R-loop studies in plants. We then focus on recent advances in the functional characterization of R-loops in Arabidopsis thaliana and rice. Recent studies in plants indicate that there are coordinated relationships between R-loops and gene expression, and between R-loops and epigenomic signatures that depend, in part, on the types of R-loops involved. Finally, we discuss the emerging roles of R-loops in plants and directions for future research., (© The Author(s) 2021. Published by Oxford University Press on behalf of the Society for Experimental Biology. All rights reserved. For permissions, please email: journals.permissions@oup.com.)

We conducted genome-wide identification of R-loops followed by integrative analyses of R-loops with relation to gene expression and epigenetic signatures in the rice genome. We found that the correlation between gene expression levels and profiled R-loop peak levels was dependent on the positions of R-loops within gene structures (hereafter named "genic position"). Both antisense only (ASO)-R-loops and sense/antisense (S/AS)-R-loops sharply peaked around transcription start sites (TSSs), and these peak levels corresponded positively with transcript levels of overlapping genes. In contrast, sense only (SO)-R-loops were generally spread over the coding regions, and their peak levels corresponded inversely to transcript levels of overlapping genes. In addition, integrative analyses of R-loop data with existing RNA-seq, chromatin immunoprecipitation sequencing (ChIP-seq), DNase I hypersensitive sites sequencing (DNase-seq), and whole-genome bisulfite sequencing (WGBS or BS-seq) data revealed interrelationships and intricate connections among R-loops, gene expression, and epigenetic signatures. Experimental validation provided evidence that the demethylation of both DNA and histone marks can influence R-loop peak levels on a genome-wide scale. This is the first study in plants that reveals novel functional aspects of R-loops, their interrelations with epigenetic methylation, and roles in transcriptional regulation., (© 2019 Fang et al.; Published by Cold Spring Harbor Laboratory Press.)

The elucidation of epigenetic responses of salt-responsive genes facilitates understanding of the underlying mechanisms that confer salt tolerance in rice. However, it is still largely unknown how epigenetic mechanisms are associated with the expression of salt-responsive genes in rice and other crops. In this study, we reported tissue-specific gene expression and tissue-specific changes in chromatin modifications or signatures between seedlings and roots in response to salt treatment. Our study indicated that among six of individual mark examined (H3K4me3, H3K27me3, H4K12ac, H3K9ac, H3K27ac and H3K36me3), a positive association between salt-related changes in histone marks and the expression of differentially expressed genes (DEGs) was observed only for H3K9ac and H4K12ac in seedlings and H3K36me3 in roots. In contrast, chromatin states (CSs) with combinations of six histone modification marks played crucial roles in the differential expression of salt-responsive genes between seedlings and roots. Most importantly, CS7 containing the bivalent marks H3K4me3 and H3K27me3, with a mutual exclusion of functions with each other, displayed distinct functions in the expression of DEGs in both tissues. Specifically, H3K27me3 in CS7 mainly suppressed the expression of DEGs in roots, while H3K4me3 affected the expression of down- and up-regulated genes, possibly by antagonizing the repressive role of H3K27me3 in seedlings. Our findings indicate distinct impacts of the CSs on the differential expression of salt-responsive genes between seedlings and roots in rice, which provides an important background for understanding chromatin-based epigenetic mechanisms that might confer salt tolerance in plants., (� The Author(s) 2019. Published by Oxford University Press on behalf of Japanese Society of Plant Physiologists. All rights reserved. For permissions, please email: journals.permissions@oup.com.)

Recombination plays an integral role in the creation of novel genetic variation in sexually reproducing species. Despite this important role, the determinants and evolution of crossover hotspots have remained poorly understood in plants. Here, we present a comparative analysis of two rice ( Oryza sativa ) historical recombination maps from two subspecies ( indica and japonica ) using 150 resequenced genomes. Fine-scale recombination rates and crossover hotspots were validated by comparison with a consensus genetic map and empirically derived crossovers, respectively. Strikingly, nearly 80% of crossover hotspots were unique to each subspecies, despite their relatively recent divergence and broad-scale correlated recombination rates. Crossover hotspots were enriched with Stowaway and P instability factor (PIF)/Harbinger transposons and overlapped accessible chromatin regions. Increased nucleotide diversity and signatures of population differentiation augmented by Stowaway and PIF/Harbinger transposons were prevalent at subspecies-specific crossover hotspots. Motifs derived from lineage-specific indica and japonica crossover hotspots were nearly identical in the two subspecies, implicating a core set of crossover motifs in rice. Finally, Stowaway and PIF/Harbinger transposons were associated with stabilized G/C bias within highly active hotspots, suggesting that hotspot activity can be fueled by de novo variation. These results provide evolutionary insight into historical crossover hotspots as potentially powerful drivers of sequence and subspecies evolution in plants., (© 2019 American Society of Plant Biologists. All rights reserved.)

Lysine crotonylation (Kcr) is a newly discovered posttranslational modification (PTM) existing in mammals. A global crotonylome analysis was undertaken in rice ( Oryza sativa L. japonica ) using high accuracy nano-LC-MS/MS in combination with crotonylated peptide enrichment. A total of 1,265 lysine crotonylation sites were identified on 690 proteins in rice seedlings. Subcellular localization analysis revealed that 51% of the crotonylated proteins identified were localized in chloroplasts. The photosynthesis-associated proteins were also mostly enriched in total crotonylated proteins. In addition, a genomic localization analysis of histone Kcr by ChIP-seq was performed to assess the relevance between histone Kcr and the genome. Of the 10,923 identified peak regions, the majority (86.7%) of the enriched peaks were located in gene body, especially exons. Furthermore, the degree of histone Kcr modification was positively correlated with gene expression in genic regions. Compared with other published histone modification data, the Kcr was co-located with the active histone modifications. Interestingly, histone Kcr-facilitated expression of genes with existing active histone modifications. In addition, 77% of histone Kcr modifications overlapped with DNase hypersensitive sites (DHSs) in intergenic regions of the rice genome and might mark other cis-regulatory DNA elements that are different from IPA1, a transcription activator in rice seedlings. Overall, our results provide a comprehensive understanding of the biological functions of the crotonylome and new active histone modification in transcriptional regulation in plants., (© 2018 Liu et al.)

Acquisition and rearrangement of host genes by transposable elements (TEs) is an important mechanism to increase gene diversity as exemplified by the ∼3000 Pack-Mutator-like TEs in the rice genome which have acquired gene sequences (Pack-MULEs), yet remain enigmatic. To identify signatures of functioning Pack-MULEs and Pack-MULE evolution, we generated transcriptome, translatome, and epigenome datasets and compared Pack-MULEs to genes and other TE families. Approximately 40% of Pack-MULEs were transcribed with 9% having translation evidence, clearly distinguishing them from other TEs. Pack-MULEs exhibited a unique expression profile associated with specificity in reproductive tissues that may be associated with seed traits. Expressed Pack-MULEs resemble regular protein-coding genes as exhibited by a low level of DNA methylation, association with active histone marks and DNase I hypersensitive sites, and an absence of repressive histone marks, suggesting that a substantial fraction of Pack-MULEs are potentially functional in vivo. Interestingly, the expression capacity of Pack-MULEs is independent of the local genomic environment, and the insertion and expression of Pack-MULEs may have altered the local chromosomal expression pattern as well as counteracted the impact of recombination on chromosomal base composition, which has profound consequences on the evolution of chromosome structure.

The precise positioning of nucleosomes along the underlying DNA is critical for a variety of biological processes, especially in regulating transcription. The interplay between nucleosomes and transcription factors for accessing the underlying DNA sequences is one of the key determinants that affect transcriptional regulation. Moreover, nucleosomes with various packing statuses confer distinct functions in regulating gene expressions in response to various internal or external signals. Therefore, global mapping of nucleosome positions is one informative way to elucidate the relationship between patterns of nucleosome positioning/occupancy and transcriptional regulations. MNase digestion coupled with high-throughput sequencing (MNase-seq) has been utilized widely for global mapping of nucleosome positioning in eukaryotes that have a sequenced genome. We have developed a robust MNase-seq procedure in plants. It mainly includes plant nuclei isolation, treatment of purified nuclei with MNase, gel recovery of MNase-trimmed mononucleosomal DNA with an approximate size of 150 bp, MNase-seq library preparation followed by Illumina sequencing, and data analysis. MNase-seq has already been successfully applied to identify genome-wide nucleosome positioning in model plants, rice, and Arabidopsis thaliana.

Background: The role of histone modifications in the DNA damage response has been extensively studied in non-plant systems, including mammals and yeast. However, there is a lack of detailed evidence showing how chromatin dynamics, either an individual mark or combined chromatin states, participate in regulating differentially expressed genes in the plant DNA damage response., Results: In this study, we used RNA-seq and ChIP-seq to show that differentially expressed genes (DEGs), in response to ionizing radiation (IR), might be involved in different pathways responsible for the DNA damage response. Moreover, chromatin structures associated with promoters, exons and intergenic regions are significantly affected by IR. Most importantly, either an individual mark or a certain chromatin state was found to be highly correlated with the expression of up-regulated genes. In contrast, only the chromatin states, as opposed to any individual marks tested, are related to the expression of the down-regulated genes., Conclusions: Our findings demonstrate that IR-related DEGs are modulated by distinct epigenetic mechanisms. Either chromatin states or distinct histone dynamics may act sequentially or in combination in regulating up-regulated genes, but the complex chromatin structure is mainly responsible for the expression of down-regulated genes. Thus, this study provides new insights into how up- and down-regulated genes are epigenetically regulated at the chromatin levels, thereby helping us to understand distinct epigenetic mechanisms that function in the plant DNA damage response.

As a histone variant, H2A.Z is highly conserved among species and plays a significant role in diverse cellular processes. Here, we generated genome-wide maps of H2A.Z in Oryza sativa (rice) callus and seedling by combining chromatin immunoprecipitation using H2A.Z antibody and high-throughput sequencing. We found a significantly high peak and a small peak of H2A.Z distributed at the 5' and 3' ends of highly expressed genes, respectively. H2A.Z was also associated with inactive genes in both tissues. H3 lysine 4 trimethylation was associated with H2A.Z deposition at the 5' end of expressed genes, and H3 lysine 27 trimethylation peaks were partially associated with H2A.Z. In summary, our study provides global analysis data for the distribution of H2A.Z in the rice genome. Our results demonstrate that the differential deposition of H2A.Z might play important roles in gene transcription during rice development., (© 2016 The Authors The Plant Journal © 2016 John Wiley & Sons Ltd.)

Background: Bidirectional gene pairs are highly abundant and mostly co-regulated in eukaryotic genomes. The structural features of bidirectional promoters (BDPs) have been well studied in yeast, humans and plants. However, the underlying mechanisms responsible for the coexpression of BDPs remain understudied, especially in plants., Results: Here, we characterized chromatin features associated with rice BDPs. Several unique chromatin features were present in rice BDPs but were missing from unidirectional promoters (UDPs), including overrepresented active histone marks, canonical nucleosomes and underrepresented H3K27me3. In particular, overrepresented active marks (H3K4ac, H4K12ac, H4K16ac, H3K4me2 and H3K36me3) were truly overrepresented in type I BDPs but not in the other two BDPs, based on a Kolmogorov-Smirnov test., Conclusions: Our analyses indicate that active marks (H3K4ac, H4K12ac, H4K16ac, H3K4me3, H3K9ac and H3K27ac) may coordinate with repressive marks (H3K27me3 and H3K9me1/3) to build a unique chromatin structure that favors the coregulation of bidirectional gene pairs. Thus, our findings help to enhance the understanding of unique epigenetic mechanisms that regulate bidirectional gene pairs and may improve the manipulation of gene pairs for crop bioengineering.

Bidirectional gene pairs tend to be highly coregulated and function in similar biological processes in eukaryotic genomes. Structural features and functional consequences of bidirectional promoters (BDPs) have received considerable attention among diverse species. However, the underlying mechanisms responsible for the bidirectional transcription and coexpression of BDPs remain poorly understood in plants. In this study, we integrated DNase-seq, RNA-seq, ChIP-seq and MNase-seq data and investigated the effect of physical DNase I hypersensitive site (DHS) positions on the transcription of rice BDPs. We found that the physical position of a DHS relative to the TSS of bidirectional gene pairs can affect the expression of the corresponding genes: the closer a DHS is to the TSS, the higher is the expression level of the genes. Most importantly, we observed that the distribution of DHSs plays a significant role in the regulation of transcription and the coexpression of gene pairs, which are possibly mediated by orchestrating the positioning of histone marks and canonical nucleosomes around BDPs. Our results demonstrate that the combined actions of chromatin structures with DHSs, which contain functional cis-elements for interaction with transcriptional machinery, may play an important role in the regulation of the bidirectional transcription or coexpression in rice BDPs. Our findings may help to enhance the understanding of DHSs in the regulation of bidirectional gene pairs.

Objective: To explore the method for estimating the cumulative staple food intake of resident., Methods: A curve fitting equation based on three dietary survey data of resident in Dayu County of Jiangxi Province was established., Results: The equation were y =- 0. 2255x~2+ 152. 19x- 391. 68 for male and y =- 0. 0985x~2+110. 41x- 150. 03 for female. And the curve fitting were good( R_M~2= 0. 9989, R_F~2=0. 9992)., Conclusion: In the area that rice is the stable food, the curve equation can be used to estimate the cumulative rice consumption of resident.

Objective: To explore a more accurate method for calculating the cumulative cadmium intake from rice., Methods: The cumulative cadmium intake from rice was calculated by using the age coefficient method and the curve equation of rice consumption, and the results were compared., Results: There was little difference between the two results in male and female group. But the equation calculation values was more closer to the actual values and it was similar between different gender. But the result calculated by the age coefficient method between different gender was significantly different., Conclusion: In the area that rice is the main medium of dietary cadmium intake, there was little difference between the two methods. Using curve equation of rice consumption to calculate the cumulative cadmium intake from rice is more feasible.

The fundamental unit of chromatin is the nucleosome that consists of a protein octamer composed of the four core histones (Hs; H3, H4, H2A, and H2B) wrapped by 147 bp of DNA. Nucleosome occupancy and positioning have proven to be dynamic and have a critical impact on expression, regulation, and evolution of eukaryotic genes. We developed nucleosome occupancy and positioning data sets using leaf tissue of rice (Oryza sativa) and both leaf and flower tissues of Arabidopsis (Arabidopsis thaliana). We show that model plant and animal species share the fundamental characteristics associated with nucleosome dynamics. Only 12% and 16% of the Arabidopsis and rice genomes, respectively, were occupied by well-positioned nucleosomes. The cores of positioned nucleosomes were enriched with G/C dinucleotides and showed a lower C→T mutation rate than the linker sequences. We discovered that nucleosomes associated with heterochromatic regions were more spaced with longer linkers than those in euchromatic regions in both plant species. Surprisingly, different nucleosome densities were found to be associated with chromatin in leaf and flower tissues in Arabidopsis. We show that deep MNase-seq data sets can be used to map nucleosome occupancy of specific genomic loci and reveal gene expression patterns correlated with chromatin dynamics in plant genomes., (© 2015 American Society of Plant Biologists. All Rights Reserved.)

Nucleosome positioning dictates the DNA accessibility for regulatory proteins, and thus is critical for gene expression and regulation. It has been well documented that only a subset of nucleosomes are reproducibly positioned in eukaryotic genomes. The most prominent example of phased nucleosomes is the context of genes, where phased nucleosomes flank the transcriptional starts sites (TSSs). It is unclear, however, what factors determine nucleosome positioning in regions that are not close to genes. We mapped both nucleosome positioning and DNase I hypersensitive site (DHS) datasets across the rice genome. We discovered that DHSs located in a variety of contexts, both genic and intergenic, were flanked by strongly phased nucleosome arrays. Phased nucleosomes were also found to flank DHSs in the human genome. Our results suggest the barrier model may represent a general feature of nucleosome organization in eukaryote genomes. Specifically, regions bound with regulatory proteins, including intergenic regions, can serve as barriers that organize phased nucleosome arrays on both sides. Our results also suggest that rice DHSs often span a single, phased nucleosome, similar to the H2A.Z-containing nucleosomes observed in DHSs in the human genome.

Plant and animal centromeres comprise megabases of highly repeated satellite sequences, yet centromere function can be specified epigenetically on single-copy DNA by the presence of nucleosomes containing a centromere-specific variant of histone H3 (cenH3). We determined the positions of cenH3 nucleosomes in rice (Oryza sativa), which has centromeres composed of both the 155-bp CentO satellite repeat and single-copy non-CentO sequences. We find that cenH3 nucleosomes protect 90-100 bp of DNA from micrococcal nuclease digestion, sufficient for only a single wrap of DNA around the cenH3 nucleosome core. cenH3 nucleosomes are translationally phased with 155-bp periodicity on CentO repeats, but not on non-CentO sequences. CentO repeats have an ∼10-bp periodicity in WW dinucleotides and in micrococcal nuclease cleavage, providing evidence for rotational phasing of cenH3 nucleosomes on CentO and suggesting that satellites evolve for translational and rotational stabilization of centromeric nucleosomes.

The wild species of the genus Oryza contain a largely untapped reservoir of agronomically important genes for rice improvement. Here we report the 261-Mb de novo assembled genome sequence of Oryza brachyantha. Low activity of long-terminal repeat retrotransposons and massive internal deletions of ancient long-terminal repeat elements lead to the compact genome of Oryza brachyantha. We model 32,038 protein-coding genes in the Oryza brachyantha genome, of which only 70% are located in collinear positions in comparison with the rice genome. Analysing breakpoints of non-collinear genes suggests that double-strand break repair through non-homologous end joining has an important role in gene movement and erosion of collinearity in the Oryza genomes. Transition of euchromatin to heterochromatin in the rice genome is accompanied by segmental and tandem duplications, further expanded by transposable element insertions. The high-quality reference genome sequence of Oryza brachyantha provides an important resource for functional and evolutionary studies in the genus Oryza.

Gene expression is controlled by the complex interaction of transcription factors binding to promoters and other regulatory DNA elements. One common characteristic of the genomic regions associated with regulatory proteins is a pronounced sensitivity to DNase I digestion. We generated genome-wide high-resolution maps of DNase I hypersensitive (DH) sites from both seedling and callus tissues of rice (Oryza sativa). Approximately 25% of the DH sites from both tissues were found in putative promoters, indicating that the vast majority of the gene regulatory elements in rice are not located in promoter regions. We found 58% more DH sites in the callus than in the seedling. For DH sites detected in both the seedling and callus, 31% displayed significantly different levels of DNase I sensitivity within the two tissues. Genes that are differentially expressed in the seedling and callus were frequently associated with DH sites in both tissues. The DNA sequences contained within the DH sites were hypomethylated, consistent with what is known about active gene regulatory elements. Interestingly, tissue-specific DH sites located in the promoters showed a higher level of DNA methylation than the average DNA methylation level of all the DH sites located in the promoters. A distinct elevation of H3K27me3 was associated with intergenic DH sites. These results suggest that epigenetic modifications play a role in the dynamic changes of the numbers and DNase I sensitivity of DH sites during development.

The presence of the centromere-specific histone H3 variant, CENH3, defines centromeric (CEN) chromatin, but poorly understood epigenetic mechanisms determine its establishment and maintenance. CEN chromatin is embedded within pericentromeric heterochromatin in most higher eukaryotes, but, interestingly, it can show euchromatic characteristics; for example, the euchromatic histone modification mark dimethylated H3 Lys 4 (H3K4me2) is uniquely associated with animal centromeres. To examine the histone marks and chromatin properties of plant centromeres, we developed a genomic tiling array for four fully sequenced rice (Oryza sativa) centromeres and used chromatin immunoprecipitation-chip to study the patterns of four euchromatic histone modification marks: H3K4me2, trimethylated H3 Lys 4, trimethylated H3 Lys 36, and acetylated H3 Lys 4, 9. The vast majority of the four histone marks were associated with genes located in the H3 subdomains within the centromere cores. We demonstrate that H3K4me2 is not a ubiquitous component of rice CEN chromatin, and the euchromatic characteristics of rice CEN chromatin are hallmarks of the transcribed sequences embedded in the centromeric H3 subdomains. We propose that the transcribed sequences located in rice centromeres may provide a barrier preventing loading of CENH3 into the H3 subdomains. The separation of CENH3 and H3 subdomains in the centromere core may be favorable for the formation of three-dimensional centromere structure and for rice centromere function.

We conducted genome-wide mapping of cytosine methylation using methylcytosine immunoprecipitation combined with Illumina sequencing. The chromosomal distribution pattern of methylated DNA is similar to the heterochromatin distribution pattern on rice chromosomes. The DNA methylation patterns of rice genes are similar to those in Arabidopsis thaliana, including distinct methylation patterns asssociated with gene bodies and promoters. The DNA sequences in the core domains of rice Cen4, Cen5 and Cen8 showed elevated methylation levels compared with sequences in the pericentromeric regions. In addition, elevated methylation levels were associated with the DNA sequences in the CENH3-binding subdomains, compared with the sequences in the flanking H3 subdomains. In contrast, the centromeric domain of Cen11, which is composed exclusively of centromeric satellite DNA, is hypomethylated compared with the pericentromeric domains. Thus, the DNA sequences associated with functional centromeres can be either hypomethylated or hypermethylated. The methylation patterns of centromeric DNA appear to be correlated with the composition of the associated DNA sequences. We propose that both hypomethylation and hypermethylation of CENH3-associated DNA sequences can serve as epigenetic marks to distinguish where CENH3 deposition will occur within the surrounding H3 chromatin., (© 2010 The Authors. Journal compilation © 2010 Blackwell Publishing Ltd.)

Most eukaryotic centromeres contain large quantities of repetitive DNA, such as satellite repeats and retrotransposons. Unlike most transposons in plant genomes, the centromeric retrotransposon (CR) family is conserved over long evolutionary periods among a majority of the grass species. CR elements are highly concentrated in centromeres, and are likely to play a role in centromere function. In order to study centromere evolution in the Oryza (rice) genus, we sequenced the orthologous region to centromere 8 of Oryza sativa from a related species, Oryza brachyantha. We found that O. brachyantha does not have the canonical CRR (CR of rice) found in the centromeres of all other Oryza species. Instead, a new Ty3-gypsy (Metaviridae) retroelement (FRetro3) was found to colonize the centromeres of this species. This retroelement is found in high copy numbers in the O. brachyantha genome, but not in other Oryza genomes, and based on the dating of long terminal repeats (LTRs) of FRetro3 it was amplified in the genome in the last few million years. Interestingly, there is a high level of removal of FRetro3 based on solo-LTRs to full-length elements, and this rapid turnover may have played a role in the replacement of the canonical CRR with the new element by active deletion. Comparison with previously described ChIP cloning data revealed that FRetro3 is found in CENH3-associated chromatin sequences. Thus, within a single lineage of the Oryza genus, the canonical component of grass centromeres has been replaced with a new retrotransposon that has all the hallmarks of a centromeric retroelement.

Objective: To observe rice cadmium level in a contaminated area where has being continuously polluted for 19 years since last investigation., Methods: Samples of late rice (polished) grown in 2006 were collected from farmer household in contaminated and control area and its cadmium concentration were detected with IPC-MS., Results: Samples of polished rice from the polluted and control areas were detected. The average cadmium concentration from last investigation area 19 years before was 0.59 mg/kg (0.05 mg/kg for rice from control area), it upraises about 30% corresponding to average of rice from the same areas detected before 19 years. The average cadmium level of rice from whole contaminated areas (including last investigation and expanding areas) was 0.26 mg/kg and it is greater than that in control areas (0.06 mg/kg) and the limited value of national hygiene standard (< 0.20 mg/kg). There was about 64%, 23.17% and 11% of samples have higher level than 0.20 mg/kg (the limited value of national hygiene standard), 0.4 mg/kg (the limited value of Codex General Standard) and 1.0 mg/kg (cadmium-rice) respectively. There was a higher percentage (36.8%) of Cadmium-rice (greater than 1.0 mg/kg) in some heavy contamination villages., Conclusion: Environmental cadmium pollution has being existing for 19 years since last investigation. Average cadmium concentration of rice from whole country polluted areas was higher than that of national hygiene standard. Percentage of "cadmium-rice" might be higher in some villages.

Paracentric inversion is known to inhibit genetic recombination between normal and inverted chromosomal segments in heterozygous arrangements. Insect inversion polymorphisms have been studied to reveal adaptive processes for maintaining genetic variation. We report the first paracentric inversion in rice (Oryza sativa), which was discovered in our effort to clone the floral organ number gene FON3. Recombination at the FON3 locus on the long arm of chromosome 11 was severely suppressed over a distance of more than 36 cM. An extensive screening among 8,242 F(2) progeny failed to detect any recombinants. Cytological analysis revealed a loop-like structure on pachytene chromosomes, whereas FISH analysis showed the migration of a BAC clone from a distal location to a position closer to the centromere. Interestingly, the locations where the genetic recombination suppression began were coincided with the positions of two physical gaps on the chromosome 11, suggesting a correlation between the physical gaps, the inversion breakpoints. Transposons and retrotransposons, and tandemly arranged members of gene families were among the sequences immediately flanking the gaps. Taken together, we propose that the genetic suppression at the FON3 locus was caused by a paracentric inversion. The possible genetic mechanism causing such a spontaneous inversion was proposed.

Anther culture offers a rapid method of generating homozygous lines for breeding program and genetic analysis. To produce homozygous transgenic lines of rice (Oryza sativa L.) in one step, we developed an efficient protocol of anther-callus-based transformation mediated by Agrobacterium after optimizing several factors influencing efficient transformation, including callus induction and Agrobacterium density for co-cultivation. Using this protocol, we obtained 145 independent green transformants from five cultivars of japonica rice by transformation with a binary vector pCXK1301 bearing the rice gene, Xa21 for resistance to bacterial blight, of which 140 were further confirmed by PCR and Southern hybridization analysis, including haploids (32.1%), diploids (62.1%) and mixoploids (7.5%). Fifteen diploids were found to be doubled haploids, which accounted for 10.7% of the total positive lines. Finally, by including 28 from colchicine induced or spontaneous diploidization of haploids later after transformation, a total of 43 doubled haploids (30.7%) of Xa21 transgenic lines were obtained. We also generated two RNAi transgenic haploids of the rice OsMADS2 gene, a putative redundant gene of OsMADS4 based on their sequence similarity, to investigate its possible roles in rice flower development by this method. Flowers from the two OsMADS2 RNAi transgenic haploids displayed obvious homeotic alternations, in which lodicules were transformed into palea/lemma-like tissues, whereas identities of other floral organs were maintained. The phenotypic alternations were proved to result from specific transcriptional suppression of OsMADS2 gene by the introduced RNAi transgene. The results confirmed that OsMADS2 is involved in lodicule development of rice flower and functionally redundant with OsMADS4 gene. Our results demonstrated that rice anther culture could be adapted to gene transformation and RNAi analysis in rice.

Oryza officinalis (CC, 2n = 24) and Oryza rhizomatis (CC, 2n = 24) belong to the Oryza genus, which contains more than 20 identified wild rice species. Although much has been known about the molecular composition and organization of centromeres in Oryza sativa, relatively little is known of its wild relatives. In the present study, we isolated and characterized a 126-bp centromeric satellite (CentO-C) from three bacterial artificial chromosomes of O. officinalis. In addition to CentO-C, low abundance of CentO satellites is also present in O. officinalis. In order to determine the chromosomal locations and distributions of CentO-C (126-bp), CentO (155 bp) and TrsC (366 bp) satellite within O. officinalis, fluorescence in situ hybridization examination was done on pachytene or metaphase I chromosomes. We found that only ten centromeres (excluding centromere 7 and 2) contain CentO-C arrays in O. officinalis, while centromere 7 comprises CentO satellites, and centromere 2 is devoid of any detectable satellites. For TrsC satellites, it was detected at multiple subtelomeric regions in O. officinalis, however, in O. rhizomatis, TrsC sequences were detected both in the four centromeric regions (CEN 3, 4, 10, 11) and the multiple subtelomeric regions. Therefore, these data reveal the evolutionary diversification pattern of centromere DNA within/or between close related species, and could provide an insight into the dynamic evolutionary processes of rice centromere.

Centromeres are required for faithful segregation of chromosomes in cell division. It is not clear what kind of sequences act as functional centromeres and how centromere sequences are organized in Oryza punctata, a BB genome species. In this study, we found that the CentO centromeric satellites in O. punctata share high homology with the CentO satellites in O. sativa. The O. punctata centromeres are characterized by megabase tandem arrays that are flanked by centromere-specific retrotransposons. Immunostaining with an antibody specific to CENH3 indicates that the 165-bp CentO satellites are the major component for functional centromeres. Moreover, both strands of CentO satellites are highly methylated and transcribed and produce small interfering RNA, which may be important for the maintenance of centromeric heterochromatin and centromere function.

The functional centromeres of rice (Oryza sativa, AA genome) chromosomes contain two key DNA components: the CRR centromeric retrotransposons and a 155-bp satellite repeat, CentO. However, several wild Oryza species lack the CentO repeat. We developed a chromatin immunoprecipitation-based technique to clone DNA fragments derived from chromatin containing the centromeric histone H3 variant CenH3. Chromatin immunoprecipitation cloning was carried out in the CentO-less species Oryza rhizomatis (CC genome) and Oryza brachyantha (FF genome). Three previously uncharacterized genome-specific satellite repeats, CentO-C1, CentO-C2, and CentO-F, were discovered in the centromeres of these two species. An 80-bp DNA region was found to be conserved in CentO-C1, CentO, and centromeric satellite repeats from maize and pearl millet, species which diverged from rice many millions of years ago. In contrast, the CentO-F repeat shows no sequence similarity to other centromeric repeats but has almost completely replaced other centromeric sequences in O. brachyantha, including the CRR-related sequences that normally constitute a significant fraction of the centromeric DNA in grass species.