CHH Context Research Articles

The effect of RNA polymerase V on 24-nt siRNA accumulation depends on DNA methylation contexts and histone modifications in rice

Significance Rice RNA polymerase V (Pol V) influences the accumulation of 24-nucleotide small interfering RNAs (24-nt siRNAs) in a locus-specific manner. Accumulation of 24-nt siRNAs is dependent on Pol V on loci with high CHH methylation levels, low CG and CHG methylation levels, and high active histone modifications. In contrast, a low methylation level in the CHH context, high methylation levels in CG and CHG contexts, and enrichment of repressive histone modifications predispose 24-nt siRNA accumulation to be independent of Pol V. DNA methylation is essential for 24-nt siRNA biosynthesis on Pol V–dependent loci but not on Pol V–independent loci. Our results suggest that monocot Pol V–transcribed scaffold RNAs are essential for reproductive development and 24-nt siRNA biosynthesis on euchromatin.

GSNOR Contributes to Demethylation and Expression of Transposable Elements and Stress-Responsive Genes.

In the past, reactive nitrogen species (RNS) were supposed to be stress-induced by-products of disturbed metabolism that cause oxidative damage to biomolecules. However, emerging evidence demonstrates a substantial role of RNS as endogenous signals in eukaryotes. In plants, S-nitrosoglutathione (GSNO) is the dominant RNS and serves as the •NO donor for S-nitrosation of diverse effector proteins. Remarkably, the endogenous GSNO level is tightly controlled by S-nitrosoglutathione reductase (GSNOR) that irreversibly inactivates the glutathione-bound NO to ammonium. Exogenous feeding of diverse RNS, including GSNO, affected chromatin accessibility and transcription of stress-related genes, but the triggering function of RNS on these regulatory processes remained elusive. Here, we show that GSNO reductase-deficient plants (gsnor1-3) accumulate S-adenosylmethionine (SAM), the principal methyl donor for methylation of DNA and histones. This SAM accumulation triggered a substantial increase in the methylation index (MI = [SAM]/[S-adenosylhomocysteine]), indicating the transmethylation activity and histone methylation status in higher eukaryotes. Indeed, a mass spectrometry-based global histone profiling approach demonstrated a significant global increase in H3K9me2, which was independently verified by immunological detection using a selective antibody. Since H3K9me2-modified regions tightly correlate with methylated DNA regions, we also determined the DNA methylation status of gsnor1-3 plants by whole-genome bisulfite sequencing. DNA methylation in the CG, CHG, and CHH contexts in gsnor1-3 was significantly enhanced compared to the wild type. We propose that GSNOR1 activity affects chromatin accessibility by controlling the transmethylation activity (MI) required for maintaining DNA methylation and the level of the repressive chromatin mark H3K9me2.

DNA methylation is involved in acclimation to iron‐deficiency in rice (Oryza sativa)

Iron (Fe) is an essential micronutrient in plants, and Fe limitation significantly affects plant growth, yield and food quality. While many studies have reported the transcriptomic profile and pursue molecular mechanism in response to Fe limitation, little is known if epigenetic factors play a role in response to Fe-deficiency. In this study, whole-genome bisulfite sequencing analysis, high-throughput RNA-Seq of mRNA, small RNA and transposable element (TE) expression with root and shoot organs of rice seedlings under Fe-sufficient and Fe-deficient conditions were performed. The results showed that widespread hypermethylation, especially for the CHH context, occurred after Fe-deficiency. Integrative analysis of methylation and transcriptome revealed that the transcript abundance of Fe-deficiency-induced genes was negatively correlated with nearby TEs and positively with the 24-nucleotide siRNAs. The ability of methylation to affect the physiology and molecular response to Fe-deficiency was tested using an exogenous DNA methyltransferase inhibitor (5-azacytidine), and genetically using a mutant for domains rearranged methyltransferase 2 (DRM2), that lacks CHH methylation. Both approaches resulted in decreased growth and Fe content in rice plants. Thus, alterations in specific methylation patterns, directed by siRNAs, play an important role in acclimation of rice to Fe-deficient conditions. Furthermore, comparison with other reports suggests this may be a universal mechanism to acclimate to limited nutrient availability.

DNA methylation-mediated modulation of rapid desiccation tolerance acquisition and dehydration stress memory in the resurrection plant Boea hygrometrica.

Pre-exposure of plants to various abiotic conditions confers improved tolerance to subsequent stress. Mild drought acclimation induces acquired rapid desiccation tolerance (RDT) in the resurrection plant Boea hygrometrica, but the mechanisms underlying the priming and memory processes remain unclear. In this study, we demonstrated that drought acclimation-induced RDT can be maintained for at least four weeks but was completely erased after 18 weeks based on a combination of the phenotypic and physiological parameters. Global transcriptome analysis identified several RDT-specific rapid dehydration-responsive genes related to cytokinin and phospholipid biosynthesis, nitrogen and carbon metabolism, and epidermal morphogenesis, most of which were pre-induced by drought acclimation. Comparison of whole-genome DNA methylation revealed dehydration stress-responsive hypomethylation in the CG, CHG, and CHH contexts and acclimation-induced hypermethylation in the CHH context of the B. hygrometrica genome, consistent with the transcriptional changes in methylation pathway genes. As expected, the global promoter and gene body methylation levels were negatively correlated with gene expression levels in both acclimated and dehydrated plants but showed no association with transcriptional divergence during the procedure. Nevertheless, the promoter methylation variations in the CG and CHG contexts were significantly associated with the differential expression of genes required for fundamental genetic processes of DNA conformation, RNA splicing, translation, and post-translational protein modification during acclimation, growth, and rapid dehydration stress response. It was also associated with the dehydration stress-induced upregulation of memory genes, including pre-mRNA-splicing factor 38A, vacuolar amino acid transporter 1-like, and UDP-sugar pyrophosphorylase, which may contribute directly or indirectly to the improvement of dehydration tolerance in B. hygrometrica plants. Altogether, our findings demonstrate the potential implications of DNA methylation in dehydration stress memory and, therefore, provide a molecular basis for enhanced dehydration tolerance in plants induced by drought acclimation.

Tissue and/or developmental stage specific methylation of nrDNA in Capsicum annuum.

The nuclear ribosomal DNA (nrDNA) sequences are often used for phylogenetic analysis among organisms. Because DNA cytosine methylation and nucleolar dominancy are two common epigenetic mechanisms of nrDNA, we hypothesized that internal transcribed spacer 1 (ITS1), 5.8S rRNA and ITS2 of nrDNA sequences could be used as epigenetic biomarkers. Thus, this research was undertaken to study level and pattern of site-specific cytosine methylation of ITS1, 5.8S and ITS2 in nine tissues and/or developmental stage of pepper Capsicum annuum L. cultivar Demre Sivrisi. Tissues studied consisted of young and old roots at 30 and 90days after sowing (das), mature dry seeds and seeds at 26days of post anthesis (dpa), flowering buds at 1day before flowering, pericarps at 3, 15 and 65 dpa. Levels and patterns of DNA cytosine methylation were identified at single base resolution using bisulfite conversion sequencing. Results of this study revealed that DNA cytosine level and pattern of ITS1, 5.8S and ITS2 were different in most tissues and/or developmental stages studied. In addition, methylation levels of CG, CHG and CHH contexts were also significantly different among the regions. Based on the findings of this study, it was concluded that high level of methylation of nrDNA sequences was relatively higher as observed in transposable element and promoter. On the other hand, its tissue-specific gene expression was effective as that of gene body and promoter methylation. Overall findings revealed that methylation levels of nrDNA could be used as biomarkers for tissue identification or age estimation in plants.

Differential Epigenetic Marks Are Associated with Apospory Expressivity in Diploid Hybrids of Paspalum rufum.

Apomixis seems to emerge from the deregulation of preexisting genes involved in sexuality by genetic and/or epigenetic mechanisms. The trait is associated with polyploidy, but diploid individuals of Paspalum rufum can form aposporous embryo sacs and develop clonal seeds. Moreover, diploid hybrid families presented a wide apospory expressivity variation. To locate methylation changes associated with apomixis expressivity, we compare relative DNA methylation levels, at CG, CHG, and CHH contexts, between full-sib P. rufum diploid genotypes presenting differential apospory expressivity. The survey was performed using a methylation content-sensitive enzyme ddRAD (MCSeEd) strategy on samples at premeiosis/meiosis and postmeiosis stages. Based on the relative methylation level, principal component analysis and heatmaps, clearly discriminate samples with contrasting apospory expressivity. Differential methylated contigs (DMCs) showed 14% of homology to known transcripts of Paspalum notatum reproductive transcriptome, and almost half of them were also differentially expressed between apomictic and sexual samples. DMCs showed homologies to genes involved in flower growth, development, and apomixis. Moreover, a high proportion of DMCs aligned on genomic regions associated with apomixis in Setaria italica. Several stage-specific differential methylated sequences were identified as associated with apospory expressivity, which could guide future functional gene characterization in relation to apomixis success at diploid and tetraploid levels.

Title: Hypermethylation of miRNA Genes During Nodule Development.

DNA methylation has recently emerged as a powerful regulatory mechanism controlling the expression of key regulators of various developmental processes, including nodulation. However, the functional role of DNA methylation in regulating the expression of microRNA (miRNA) genes during the formation and development of nitrogen-fixing nodules remains largely unknown. In this study, we profiled DNA methylation patterns of miRNA genes during nodule formation, development, and early senescence stages in soybean (Glycine max) through the analysis of methylC—seq data. Absolute DNA methylation levels in the CG, CHH, and CHH sequence contexts over the promoter and primary transcript regions of miRNA genes were significantly higher in the nodules compared with the corresponding root tissues at these three distinct nodule developmental stages. We identified a total of 82 differentially methylated miRNAs in the nodules compared with roots. Differential DNA methylation of these 82 miRNAs was detected only in the promoter (69), primary transcript region (3), and both in the promoter and primary transcript regions (10). The large majority of these differentially methylated miRNAs were hypermethylated in nodules compared with the corresponding root tissues and were found mainly in the CHH context and showed stage-specific methylation patterns. Differentially methylated regions in the promoters of 25 miRNAs overlapped with transposable elements, a finding that may explain the vulnerability of miRNAs to DNA methylation changes during nodule development. Gene expression analysis of a set of promoter-differentially methylated miRNAs pointed to a negative association between DNA methylation and miRNA expression. Gene Ontology and pathways analyses indicate that changes in DNA methylation of miRNA genes are reprogrammed and contribute to nodule development through indirect regulation of genes involved in cellular processes and pathways with well-established roles in nodulation.

Identification of epigenetic variation associated with synchronous pod maturity in mungbean (Vigna radiata L.)

Cytosine methylation in genomic DNA affects gene expression, potentially causing phenotypic variation. Mungbean, an agronomically and nutritionally important legume species, is characterized by nonsynchronous pod maturity, resulting in multiple harvest which costs extra time and labor. To elucidate the epigenetic influences on synchronous pod maturity (SPM) in mungbean, we determined the genome-wide DNA methylation profiles of eight mungbean recombinant inbred lines (RILs) and their parental genotypes, and compared DNA methylation profiles between high SPM and low SPM RILs, thus revealing differentially methylated regions (DMRs). A total of 3, 18, and 28 pure DMRs, defined as regions showing no significant correlation between nucleotide sequence variation and methylation level, were identified in CpG, CHG, and CHH contexts, respectively. These DMRs were proximal to 20 genes. Among the 544 single nucleotide polymorphisms identified near the 20 genes, only one caused critical change in gene expression by early termination. Analysis of these genome-wide DNA methylation profiles suggests that epigenetic changes can influence the expression of proximal genes, regardless of nucleotide sequence variation, and that SPM is mediated through gibberellin-mediated hormone signaling pathways. These results provide insights into how epialleles contribute to phenotypic variation and improve SPM in mungbean cultivars.

Efficient and accurate determination of genome-wide DNA methylation patterns in Arabidopsis thaliana with enzymatic methyl sequencing

Background5′ methylation of cytosines in DNA molecules is an important epigenetic mark in eukaryotes. Bisulfite sequencing is the gold standard of DNA methylation detection, and whole-genome bisulfite sequencing (WGBS) has been widely used to detect methylation at single-nucleotide resolution on a genome-wide scale. However, sodium bisulfite is known to severely degrade DNA, which, in combination with biases introduced during PCR amplification, leads to unbalanced base representation in the final sequencing libraries. Enzymatic conversion of unmethylated cytosines to uracils can achieve the same end product for sequencing as does bisulfite treatment and does not affect the integrity of the DNA; enzymatic methylation sequencing may, thus, provide advantages over bisulfite sequencing.ResultsUsing an enzymatic methyl-seq (EM-seq) technique to selectively deaminate unmethylated cytosines to uracils, we generated and sequenced libraries based on different amounts of Arabidopsis input DNA and different numbers of PCR cycles, and compared these data to results from traditional whole-genome bisulfite sequencing. We found that EM-seq libraries were more consistent between replicates and had higher mapping and lower duplication rates, lower background noise, higher average coverage, and higher coverage of total cytosines. Differential methylation region (DMR) analysis showed that WGBS tended to over-estimate methylation levels especially in CHG and CHH contexts, whereas EM-seq detected higher CG methylation levels in certain highly methylated areas. These phenomena can be mostly explained by a correlation of WGBS methylation estimation with GC content and methylated cytosine density. We used EM-seq to compare methylation between leaves and flowers, and found that CHG methylation level is greatly elevated in flowers, especially in pericentromeric regions.ConclusionWe suggest that EM-seq is a more accurate and reliable approach than WGBS to detect methylation. Compared to WGBS, the results of EM-seq are less affected by differences in library preparation conditions or by the skewed base composition in the converted DNA. It may therefore be more desirable to use EM-seq in methylation studies.

Genetic and Methylome Variation in Turkish Brachypodium Distachyon Accessions Differentiate Two Geographically Distinct Subpopulations.

Brachypodium distachyon (Brachypodium) is a non-domesticated model grass species that can be used to test if variation in genetic sequence or methylation are linked to environmental differences. To assess this, we collected seeds from 12 sites within five climatically distinct regions of Turkey. Seeds from each region were grown under standardized growth conditions in the UK to preserve methylated sequence variation. At six weeks following germination, leaves were sampled and assessed for genomic and DNA methylation variation. In a follow-up experiment, phenomic approaches were used to describe plant growth and drought responses. Genome sequencing and population structure analysis suggested three ancestral clusters across the Mediterranean, two of which were geographically separated in Turkey into coastal and central subpopulations. Phenotypic analyses showed that the coastal subpopulation tended to exhibit relatively delayed flowering and the central, increased drought tolerance as indicated by reduced yellowing. Genome-wide methylation analyses in GpC, CHG and CHH contexts also showed variation which aligned with the separation into coastal and central subpopulations. The climate niche modelling of both subpopulations showed a significant influence from the “Precipitation in the Driest Quarter” on the central subpopulation and “Temperature of the Coldest Month” on the coastal subpopulation. Our work demonstrates genetic diversity and variation in DNA methylation in Turkish accessions of Brachypodium that may be associated with climate variables and the molecular basis of which will feature in ongoing analyses.

Cytosine methylations in the promoter regions of genes involved in the cellular oxidation equilibrium pathways affect rice heat tolerance

BackgroundHigh temperatures, particularly at night, decrease rice yield and quality. As high nighttime temperatures (HNTs) become increasingly frequent due to climate change, it is imperative to develop rice crops that tolerate HNTs. DNA methylation may represent a potential avenue for HNT-tolerant rice strain development, as this mechanism regulates gene activity and cellular phenotype in response to adverse environmental conditions without changing the nucleotide sequence.ResultsAfter HNT exposure, the methylation patterns of cytosines in the CHH context differed noticeably between two coisogenic rice strains with significantly different levels in heat tolerance. Methylation differences between strains were primarily observed on successive cytosines in the promoter or downstream regions of transcription factors and transposon elements. In contrast to the heat-sensitive rice strain, the regions 358–359 bp and 2–60 bp downstream of two basal transcriptional factors (TFIID subunit 11 and mediator of RNA polymerase II transcription subunit 31, respectively) were fully demethylated in the heat-tolerant strain after HNT exposure. In the heat-tolerant strain, HNTs reversed the methylation patterns of successive cytosines in the promoter regions of various genes involved in abscisic acid (ABA)-related reactive oxygen species (ROS) equilibrium pathways, including the pentatricopeptide repeat domain gene PPR (LOC_Os07g28900) and the homeobox domain gene homeobox (LOC_Os01g19694). Indeed, PRR expression was inhibited in heat-sensitive rice strains, and the methylation rates of the cytosines in the promoter region of PRR were greater in heat-sensitive strains as compared to heat-tolerant strains.ConclusionsAfter HNT exposure, cytosines in the CHH context were more likely than cytosines in other contexts to be methylated differently between the heat-sensitive and heat-tolerant rice strains. Methylation in the promoter regions of the genes associated with ABA-related oxidation and ROS scavenging improved heat tolerance in rice. Our results help to clarify the molecular mechanisms underlying rice heat tolerance.

Genome-wide investigation of DNA methylation dynamics reveals a critical role of DNA demethylation during the early somatic embryogenesis of Dimocarpus longan Lour.

DNA methylation plays essential roles in gene regulation, chromatin structure stability, gene imprinting, X chromosome inactivation and embryonic development. However, the dynamics and functions of DNA methylation during the early stage of longan (Dimocarpus longan) somatic embryogenesis (SE) are still unclear. In this study, we carried out whole genome bisulphite sequencing and transcriptome sequencing analyses for embryogenic callus (EC), incomplete compact pro-embryogenic cultures (ICpEC) and globular embryos (GE) in an early SE system. At a global level, the DNA 5-methylcytosine content in EC, ICpEC and GE was 24.59, 19.65 and 19.74%, respectively, suggesting a global decrease in DNA methylation from EC to ICpEC and then a slight increase from ICpEC to GE. Differentially methylated region (DMR) analysis showed that hypomethylation mainly occurred in CHH contexts. Gene ontology and Kyoto encyclopedia of genes and genomes analysis of hypomethylated-CHH-DMR-associated genes revealed that zein biosynthesis, fatty acid biosynthesis, circadian rhythm and mitophagy pathways were involved in longan early SE. Expression patterns of DNA methyltransferase and demethylase genes during longan early SE suggested that the decrease in DNA methylation was probably regulated by DNA methyltransferase genes and the DNA demethylase gene REPRESSOR OF SILENCING 1 (ROS1). The correlation between DNA hypomethylation and gene expression revealed that decreased DNA methylation did not cause extensive changes in gene expression during early longan SE and that gene expression may be affected by methylation changes in gene and downstream regions. Inhibiting DNA methylation with 5-azacytidine treatment in EC promoted the formation of GE and enhanced the capability of longan SE. Our results suggest that DNA demethylation has important roles in longan SE development.

Retro-Element Gypsy-163 Is Differentially Methylated in Reproductive Tissues of Apomictic and Sexual Plants of Cenchrus ciliaris.

Apomixis, an asexual mode of reproduction through seeds, has immense scope for crop improvement due to its ability to fix hybrid vigor. In C. ciliaris, a predominantly apomictically reproducing range grass, apomixis is genetically controlled by an apospory-specific-genomic-region (ASGR) which is enriched with retrotransposons. Earlier studies showed insertional polymorphisms of a few ASGR-specific retrotransposons between apomictic and sexual plants of C. ciliaris. REs are mainly regulated at the transcriptional level through cytosine methylation. To understand the possible association of ASGR-specific retrotransposon to apomixis, the extent and pattern of differential methylation of Gy163 RE and its impact on transcription were investigated in two genotypes each of apomictic and sexual plants of C. ciliaris. We observed that Gy163 encodes for an integrase domain of RE Ty3-Gypsy, is differentially methylated between reproductive tissues of apomictic and sexual plants. However, leaf tissues did not exhibit differential methylation between apomictic and sexual plants. Among the three contexts (CG, CHG, and CHH) of cytosine methylation, the maximum variation was observed in CHH context in reproductive (at aposporous initial and mature embryo sac stages) tissues of apomictic plants implicating RdDM pathway in methylation of Gy163. Quantitative PCR analysis showed that Gy163 transcripts are expressed more in the reproductive tissues of apomictic plants compared to that in the sexual plants, which was negatively correlated with the methylation level. Thus, the study helps in understanding the role of RE present in ASGR in epigenetic regulation of apomictic mode of reproduction in C. ciliaris.

Dormant but Active: Chilling Accumulation Modulates the Epigenome and Transcriptome of Prunus avium During Bud Dormancy

Temperate deciduous fruit tree species like sweet cherry (Prunus avium) require long periods of low temperatures to trigger dormancy release and flowering. In addition to sequence-based genetic diversity, epigenetic variation may contribute to different chilling requirements among varieties. For the low chill variety ‘Royal Dawn’ and high chill variety ‘Kordia’, we studied the methylome of floral buds during chilling accumulation using MethylC-seq to identify differentially methylated regions (DMRs) during chilling hours (CH) accumulation, followed by transcriptome analysis to correlate changes in gene expression with DNA methylation. We found that during chilling accumulation, DNA methylation increased from 173 CH in ‘Royal Dawn’ and 443 CH in ‘Kordia’ and was mostly associated with the CHH context. In addition, transcriptional changes were observed from 443 CH in ‘Kordia’ with 1,210 differentially expressed genes, increasing to 4,292 genes at 1,295 CH. While ‘Royal Dawn’ showed approximately 5,000 genes differentially expressed at 348 CH and 516 CH, showing a reprogramming that was specific for each genotype. From conserved upregulated genes that overlapped with hypomethylated regions and downregulated genes that overlapped with hypermethylated regions in both varieties, we identified genes related to cold-sensing, cold-signaling, oxidation-reduction process, metabolism of phenylpropanoids and lipids, and a MADS-box SVP-like gene. As a complementary analysis, we used conserved and non-conserved DEGs that presented a negative correlation between DNA methylations and mRNA levels across all chilling conditions, obtaining Gene Ontology (GO) categories related to abiotic stress, metabolism, and oxidative stress. Altogether, this data indicates that changes in DNA methylation precedes transcript changes and may occur as an early response to low temperatures to increase the cold tolerance in the endodormancy period, contributing with the first methylome information about the effect of environmental cues over two different genotypes of sweet cherry.

DNA methylation reprogramming during seed development and its functional relevance in seed size/weight determination in chickpea

Seed development is orchestrated via complex gene regulatory networks and pathways. Epigenetic factors may also govern seed development and seed size/weight. Here, we analyzed DNA methylation in a large-seeded chickpea cultivar (JGK 3) during seed development stages. Progressive gain of CHH context DNA methylation in transposable elements (TEs) and higher frequency of small RNAs in hypermethylated TEs during seed development suggested a role of the RNA-dependent DNA methylation pathway. Frequency of intragenic TEs was higher in CHH context differentially methylated region (DMR) associated differentially expressed genes (DEGs). CG context hyper/hypomethylation within the gene body was observed for most of DMR-associated DEGs in JGK 3 as compared to small-seeded chickpea cultivar (Himchana 1). We identified candidate genes involved in seed size/weight determination exhibiting CG context hypermethylation within the gene body and higher expression in JGK 3. This study provides insights into the role of DNA methylation in seed development and seed size/weight determination in chickpea.

AXR1 affects DNA methylation independently of its role in regulating meiotic crossover localization.

Meiotic crossovers (COs) are important for reshuffling genetic information between homologous chromosomes and they are essential for their correct segregation. COs are unevenly distributed along chromosomes and the underlying mechanisms controlling CO localization are not well understood. We previously showed that meiotic COs are mis-localized in the absence of AXR1, an enzyme involved in the neddylation/rubylation protein modification pathway in Arabidopsis thaliana. Here, we report that in axr1-/-, male meiocytes show a strong defect in chromosome pairing whereas the formation of the telomere bouquet is not affected. COs are also redistributed towards subtelomeric chromosomal ends where they frequently form clusters, in contrast to large central regions depleted in recombination. The CO suppressed regions correlate with DNA hypermethylation of transposable elements (TEs) in the CHH context in axr1-/- meiocytes. Through examining somatic methylomes, we found axr1-/- affects DNA methylation in a plant, causing hypermethylation in all sequence contexts (CG, CHG and CHH) in TEs. Impairment of the main pathways involved in DNA methylation is epistatic over axr1-/- for DNA methylation in somatic cells but does not restore regular chromosome segregation during meiosis. Collectively, our findings reveal that the neddylation pathway not only regulates hormonal perception and CO distribution but is also, directly or indirectly, a major limiting pathway of TE DNA methylation in somatic cells.

Single-base cytosine methylation analysis in fruits of three Capsicum species

Single-base cytosine methylation analysis across fruits of Capsicum annuum, C. chinense and C. frutescens showed global average methylation ranging from 82.8–89.1%, 77.6–83.9%, and 22.4–25% at CG, CHG and CHH contexts, respectively. High gene-body methylation at CG and CHG was observed across Capsicum species. The C. annuum showed the highest proportion (>80%) of mCs at different genomic regions compared to C. chinense and C. frutescens. Cytosine methylation for transposable-elements were lower in C. frutescens compared to C. annuum and C. chinense. A total of 510,165 CG, 583112 CHG and 277,897 CHH DMRs were identified across three Capsicum species. The differentially methylated regions (DMRs) distribution analysis revealed C. frutescens as more hypo-methylated compared to C. annuum and C. chinense, and also the presence of more intergenic DMRs in Capsicum genome. At CG and CHG context, gene expression and promoter methylation showed inverse correlations. Furthermore, the observed correlation between methylation and expression of genes suggested the potential role of methylation in Capsicum fruit development/ripening.

Regulation of Leaf Longevity by DML3-Mediated DNA Demethylation

Leaf senescence is driven by the expression of senescence-associated genes (SAGs). Development-specific genes often undergo DNA demethylation in their promoter and other regions, which regulates gene expression. Whether and how DNA demethylation regulates the expression of SAGs and thus leaf senescence remain elusive. Whole-genome bisulfite sequencing (WGBS) analyses of wild-type (WT) and demeter-like 3 (dml3) Arabidopsis leaves at three developmental stages revealed hypermethylation during leaf senescence in dml3 compared with WT, and 20 556 differentially methylated regions (DMRs) were identified by comparing the methylomes of dml3 and WT in the CG, CHG, and CHH contexts. Furthermore, we identified that 335 DMR-associated genes (DMGs), such as NAC016 and SEN1, are upregulated during leaf senescence, and found an inverse correlation between the DNA methylation levels (especially in the promoter regions) and the transcript abundances of the related SAGs in WT. In contrast, in dml3 the promoters of SAGs were hypermethylated and their transcript levels were remarkably reduced, and leaf senescence was significantly delayed. Collectively, our study unraveled a novel epigenetic regulatory mechanism underlying leaf senescence in which DML3 is expressed at the onset of and during senescence to demethylate promoter, gene body or 3′ UTR regions to activate a set of SAGs.

Analysis of Global Methylome and Gene Expression during Carbon Reserve Mobilization in Stems under Soil Drying.

In rice (Oryza sativa), a specific temporary source organ, the stem, is important for grain filling, and moderate soil drying (MD) enhanced carbon reserve flow from stems to increase grain yield. The dynamics and biological relevance of DNA methylation in carbon reserve remobilization during grain filling are unknown. Here, we generated whole-genome single-base resolution maps of the DNA methylome in the stem. During grain filling under MD, we observed an increase in DNA methylation of total cytosines, with more hypomethylated than hypermethylated regions. Genes responsible for DNA methylation and demethylation were up-regulated, suggesting that DNA methylation changes in the stem were regulated by antagonism between DNA methylation and demethylation activity. In addition, methylation in the CG and CHG contexts was negatively associated with gene expression, while that in the CHH context was positively associated with gene expression. A hypermethylated/up-regulated transcription factor of MYBS2 inhibited MYB30 expression and possibly enhanced β-Amylase5 expression, promoting subsequent starch degradation in rice stems under MD treatment. In addition, a hypermethylated/down-regulated transcription factor of ERF24 was predicted to interact with, and thereby decrease the expression of, abscisic acid 8'-hydroxylase1, thus increasing abscisic acid concentration under MD treatment. Our findings provide insight into the DNA methylation dynamics in carbon reserve remobilization of rice stems, demonstrate that MD increased this remobilization, and suggest a link between DNA methylation and gene expression in rice stems during grain filling.

Machine learning analyses of methylation profiles uncovers tissue‐specific gene expression patterns in wheat

DNA methylation is a mechanism of epigenetic modification in eukaryotic organisms. Generally, methylation within genes promoter inhibits regulatory protein binding and represses transcription, whereas gene body methylation is associated with actively transcribed genes. However, it remains unclear whether there is interaction between methylation levels across genic regions and which site has the biggest impact on gene regulation. We investigated and used the methylation patterns of the bread wheat cultivar Chinese Spring to uncover differentially expressed genes (DEGs) between roots and leaves, using six machine learning algorithms and a deep neural network. As anticipated, genes with higher expression in leaves were mainly involved in photosynthesis and pigment biosynthesis processes whereas genes that were not differentially expressed between roots and leaves were involved in protein processes and membrane structures. Methylation occurred preponderantly (60%) in the CG context, whereas 35 and 5% of methylation occurred in CHG and CHH contexts, respectively. Methylation levels were highly correlated (r=0.7 to 0.9) between all genic regions, except within the promoter (r=0.4 to 0.5). Machine learning models gave a high (0.81) prediction accuracy of DEGs. There was a strong correlation (p-value=9.20×10-10 ) between all features and gene expression, suggesting that methylation across all genic regions contribute to gene regulation. However, the methylation of the promoter, the CDS and the exon in CG context was the most impactful. Our study provides more insights into the interplay between DNA methylation and gene expression and paves the way for identifying tissue-specific genes using methylation profiles.