Developmental responses of bread wheat to changes in ambient temperature following deletion of a locus that includes FLOWERING LOCUS T1

FLOWERING LOCUS T (FT) is a central integrator of environmental signals that regulates the timing of vegetative to reproductive transition in flowering plants. In model plants, these environmental signals have been shown to include photoperiod, vernalization, and ambient temperature pathways, and in crop species, the integration of the ambient temperature pathway remains less well understood. In hexaploid wheat, at least 5 FT‐like genes have been identified, each with a copy on the A, B, and D genomes. Here, we report the characterization of FT‐B1 through analysis of FT‐B1 null and overexpression genotypes under different ambient temperature conditions. This analysis has identified that the FT‐B1 alleles perform differently under diverse environmental conditions; most notably, the FT‐B1 null produces an increase in spikelet and tiller number when grown at lower temperature conditions. Additionally, absence of FT‐B1 facilitates more rapid germination under both light and dark conditions. These results provide an opportunity to understand the FT‐dependent pathways that underpin key responses of wheat development to changes in ambient temperature. This is particularly important for wheat, for which development and grain productivity are sensitive to changes in temperature

Induction of epigenetic variation in Arabidopsis by over-expression of DNA METHYLTRANSFERASE1 (MET1)

Epigenetic marks such as DNA methylation and histone modification can vary among plant accessions creating epi-alleles with different levels of expression competence. Mutations in epigenetic pathway functions are powerful tools to induce epigenetic variation. As an alternative approach, we investigated the potential of over-expressing an epigenetic function, using DNA METHYLTRANSFERASE1 (MET1) for proof-of-concept. In Arabidopsis thaliana, MET1 controls maintenance of cytosine methylation at symmetrical CG positions. At some loci, which contain dense DNA methylation in CG- and non-CG context, loss of MET1 causes joint loss of all cytosines methylation marks. We find that over-expression of both catalytically active and inactive versions of MET1 stochastically generates new epi-alleles at loci encoding transposable elements, non-coding RNAs and proteins, which results for most loci in an increase in expression. Individual transformants share some common phenotypes and genes with altered gene expression. Altered expression states can be transmitted to the next generation, which does not require the continuous presence of the MET1 transgene. Long-term stability and epigenetic features differ for individual loci. Our data show that over-expression of MET1, and potentially of other genes encoding epigenetic factors, offers an alternative strategy to identify epigenetic target genes and to create novel epi-alleles

Vernalization-Repression of Arabidopsis FLC Requires Promoter Sequences but Not Antisense Transcripts

The repression of Arabidopsis FLC expression by vernalization (extended cold) has become a model for understanding polycomb-associated epigenetic regulation in plants. Antisense and sense non-coding RNAs have been respectively implicated in initiation and maintenance of FLC repression by vernalization. We show that the promoter and first exon of the FLC gene are sufficient to initiate repression during vernalization; this initial repression of FLC does not require antisense transcription. Long-term maintenance of FLC repression requires additional regions of the gene body, including those encoding sense non-coding transcripts

Plant-RRBS, a bisulfite and next-generation sequencing-based methylome profiling method enriching for coverage of cytosine positions

Background: Cytosine methylation in plant genomes is important for the regulation of gene transcription and transposon activity. Genome-wide methylomes are studied upon mutation of the DNA methyltransferases, adaptation to environmental stresses or during development. However, from basic biology to breeding programs, there is a need to monitor multiple samples to determine transgenerational methylation inheritance or differential cytosine methylation. Methylome data obtained by sodium hydrogen sulfite (bisulfite)-conversion and next-generation sequencing (NGS) provide genome- wide information on cytosine methylation. However, a profiling method that detects cytosine methylation state dispersed over the genome would allow high-throughput analysis of multiple plant samples with distinct epigenetic signatures. We use specific restriction endonucleases to enrich for cytosine coverage in a bisulfite and NGS-based profiling method, which was compared to whole-genome bisulfite sequencing of the same plant material. Methods: We established an effective methylome profiling method in plants, termed plant-reduced representation bisulfite sequencing (plant-RRBS), using optimized double restriction endonuclease digestion, fragment end repair, adapter ligation, followed by bisulfite conversion, PCR amplification and NGS. We report a performant laboratory protocol and a straightforward bioinformatics data analysis pipeline for plant-RRBS, applicable for any reference-sequenced plant species. Results: As a proof of concept, methylome profiling was performed using an Oryza sativa ssp. indica pure breeding line and a derived epigenetically altered line (epiline). Plant-RRBS detects methylation levels at tens of millions of cytosine positions deduced from bisulfite conversion in multiple samples. To evaluate the method, the coverage of cytosine positions, the intra-line similarity and the differential cytosine methylation levels between the pure breeding line and the epiline were determined. Plant-RRBS reproducibly covers commonly up to one fourth of the cytosine positions in the rice genome when using MspI-DpnII within a group of five biological replicates of a line. The method predominantly detects cytosine methylation in putative promoter regions and not-annotated regions in rice. Conclusions: Plant-RRBS offers high-throughput and broad, genome- dispersed methylation detection by effective read number generation obtained from reproducibly covered genome fractions using optimized endonuclease combinations, facilitating comparative analyses of multi-sample studies for cytosine methylation and transgenerational stability in experimental material and plant breeding populations

DNA Methylation Causes Predominant Maternal Controls of Plant Embryo Growth

The parental conflict hypothesis predicts that the mother inhibits embryo growth counteracting growth enhancement by the father. In plants the DNA methyltransferase MET1 is a central regulator of parentally imprinted genes that affect seed growth. However the relation between the role of MET1 in imprinting and its control of seed size has remained unclear. Here we combine cytological, genetic and statistical analyses to study the effect of MET1 on seed growth. We show that the loss of MET1 during male gametogenesis causes a reduction of seed size, presumably linked to silencing of the paternal allele of growth enhancers in the endosperm, which nurtures the embryo. However, we find no evidence for a similar role of MET1 during female gametogenesis. Rather, the reduction of MET1 dosage in the maternal somatic tissues causes seed size increase. MET1 inhibits seed growth by restricting cell division and elongation in the maternal integuments that surround the seed. Our data demonstrate new controls of seed growth linked to the mode of reproduction typical of flowering plants. We conclude that the regulation of embryo growth by MET1 results from a combination of predominant maternal controls, and that DNA methylation maintained by MET1 does not orchestrate a parental conflict

Heritable Epigenetic Variation among Maize Inbreds

Epigenetic variation describes heritable differences that are not attributable to changes in DNA sequence. There is the potential for pure epigenetic variation that occurs in the absence of any genetic change or for more complex situations that involve both genetic and epigenetic differences. Methylation of cytosine residues provides one mechanism for the inheritance of epigenetic information. A genome-wide profiling of DNA methylation in two different genotypes of Zea mays (ssp. mays), an organism with a complex genome of interspersed genes and repetitive elements, allowed the identification and characterization of examples of natural epigenetic variation. The distribution of DNA methylation was profiled using immunoprecipitation of methylated DNA followed by hybridization to a high-density tiling microarray. The comparison of the DNA methylation levels in the two genotypes, B73 and Mo17, allowed for the identification of approximately 700 differentially methylated regions (DMRs). Several of these DMRs occur in genomic regions that are apparently identical by descent in B73 and Mo17 suggesting that they may be examples of pure epigenetic variation. The methylation levels of the DMRs were further studied in a panel of near-isogenic lines to evaluate the stable inheritance of the methylation levels and to assess the contribution of cis- and trans- acting information to natural epigenetic variation. The majority of DMRs that occur in genomic regions without genetic variation are controlled by cis-acting differences and exhibit relatively stable inheritance. This study provides evidence for naturally occurring epigenetic variation in maize, including examples of pure epigenetic variation that is not conditioned by genetic differences. The epigenetic differences are variable within maize populations and exhibit relatively stable trans-generational inheritance. The detected examples of epigenetic variation, including some without tightly linked genetic variation, may contribute to complex trait variation

Changes in Global Gene Expression in Response to Chemical and Genetic Perturbation of Chromatin Structure

DNA methylation is important for controlling gene expression in all eukaryotes. Microarray analysis of mutant and chemically-treated Arabidopsis thaliana seedlings with reduced DNA methylation revealed an altered gene expression profile after treatment with the DNA methylation inhibitor 5-aza-2′ deoxycytidine (5-AC), which included the upregulation of expression of many transposable elements. DNA damage-response genes were also coordinately upregulated by 5-AC treatment. In the ddm1 mutant, more specific changes in gene expression were observed, in particular for genes predicted to encode transposable elements in centromeric and pericentromeric locations. These results confirm that DDM1 has a very specific role in maintaining transcriptional silence of transposable elements, while chemical inhibitors of DNA methylation can affect gene expression at a global level

Arabidopsis RPT2a, 19S Proteasome Subunit, Regulates Gene Silencing via DNA Methylation

The ubiquitin/proteasome pathway plays a crucial role in many biological processes. Here we report a novel role for the Arabidopsis 19S proteasome subunit RPT2a in regulating gene activity at the transcriptional level via DNA methylation. Knockout mutation of the RPT2a gene did not alter global protein levels; however, the transcriptional activities of reporter transgenes were severely reduced compared to those in the wild type. This transcriptional gene silencing (TGS) was observed for transgenes under control of either the constitutive CaMV 35S promoter or the cold-inducible RD29A promoter. Bisulfite sequencing analysis revealed that both the transgene and endogenous RD29A promoter regions were hypermethylated at CG and non-CG contexts in the rpt2a mutant. Moreover, the TGS of transgenes driven by the CaMV 35S promoters was released by treatment with the DNA methylation inhibitor 5-aza-2′-deoxycytidine, but not by application of the inhibitor of histone deacetylase Trichostatin A. Genetic crosses with the DNA methyltransferase met1 single or drm1drm2cmt3 triple mutants also resulted in a release of CaMV 35S transgene TGS in the rpt2a mutant background. Increased methylation was also found at transposon sequences, suggesting that the 19S proteasome containing AtRPT2a negatively regulates TGS at transgenes and at specific endogenous genes through DNA methylation

Global Analysis of Genetic, Epigenetic and Transcriptional Polymorphisms in Arabidopsis thaliana Using Whole Genome Tiling Arrays

Whole genome tiling arrays provide a high resolution platform for profiling of genetic, epigenetic, and gene expression polymorphisms. In this study we surveyed natural genomic variation in cytosine methylation among Arabidopsis thaliana wild accessions Columbia (Col) and Vancouver (Van) by comparing hybridization intensity difference between genomic DNA digested with either methylation-sensitive (HpaII) or -insensitive (MspI) restriction enzyme. Single Feature Polymorphisms (SFPs) were assayed on a full set of 1,683,620 unique features of Arabidopsis Tiling Array 1.0F (Affymetrix), while constitutive and polymorphic CG methylation were assayed on a subset of 54,519 features, which contain a 5′CCGG3′ restriction site. 138,552 SFPs (1% FDR) were identified across enzyme treatments, which preferentially accumulated in pericentromeric regions. Our study also demonstrates that at least 8% of all analyzed CCGG sites were constitutively methylated across the two strains, while about 10% of all analyzed CCGG sites were differentially methylated between the two strains. Within euchromatin arms, both constitutive and polymorphic CG methylation accumulated in central regions of genes but under-represented toward the 5′ and 3′ ends of the coding sequences. Nevertheless, polymorphic methylation occurred much more frequently in gene ends than constitutive methylation. Inheritance of methylation polymorphisms in reciprocal F1 hybrids was predominantly additive, with F1 plants generally showing levels of methylation intermediate between the parents. By comparing gene expression profiles, using matched tissue samples, we found that magnitude of methylation polymorphism immediately upstream or downstream of the gene was inversely correlated with the degree of expression variation for that gene. In contrast, methylation polymorphism within genic region showed weak positive correlation with expression variation. Our results demonstrated extensive genetic and epigenetic polymorphisms between Arabidopsis accessions and suggested a possible relationship between natural CG methylation variation and gene expression variation

A Position Effect on the Heritability of Epigenetic Silencing

In animals and yeast, position effects have been well documented. In animals, the best example of this process is Position Effect Variegation (PEV) in Drosophila melanogaster. In PEV, when genes are moved into close proximity to constitutive heterochromatin, their expression can become unstable, resulting in variegated patches of gene expression. This process is regulated by a variety of proteins implicated in both chromatin remodeling and RNAi-based silencing. A similar phenomenon is observed when transgenes are inserted into heterochromatic regions in fission yeast. In contrast, there are few examples of position effects in plants, and there are no documented examples in either plants or animals for positions that are associated with the reversal of previously established silenced states. MuDR transposons in maize can be heritably silenced by a naturally occurring rearranged version of MuDR. This element, Muk, produces a long hairpin RNA molecule that can trigger DNA methylation and heritable silencing of one or many MuDR elements. In most cases, MuDR elements remain inactive even after Muk segregates away. Thus, Muk-induced silencing involves a directed and heritable change in gene activity in the absence of changes in DNA sequence. Using classical genetic analysis, we have identified an exceptional position at which MuDR element silencing is unstable. Muk effectively silences the MuDR element at this position. However, after Muk is segregated away, element activity is restored. This restoration is accompanied by a reversal of DNA methylation. To our knowledge, this is the first documented example of a position effect that is associated with the reversal of epigenetic silencing. This observation suggests that there are cis-acting sequences that alter the propensity of an epigenetically silenced gene to remain inactive. This raises the interesting possibility that an important feature of local chromatin environments may be the capacity to erase previously established epigenetic marks