CRWN nuclear lamina components maintain the H3K27me3 landscape and promote successful reproduction in Arabidopsis.

The nuclear lamina, a sub-nuclear protein matrix, maintains nuclear structure and genome function. Here, we investigate the role of Arabidopsis lamin analogs CROWDED NUCLEIs during gametophyte and seed development. We observed defects in crwn mutant seeds, including seed abortion and reduced germination rate. Quadruple crwn null genotypes were rarely transmitted through gametophytes. We focused on the crwn1 crwn2 (crwn1/2) endosperm, which exhibited enlarged chalazal cysts and increased expression of stress-related genes and the MADS-box transcription factor PHERES1 and its targets. Previously, it was shown that PHERES1 is regulated by H3K27me3 and that CRWN1 interacts with the PRC2 interactor PWO1. Thus, we tested whether crwn1/2 alters H3K27me3 patterns. We observed a mild loss of H3K27me3 at several hundred loci, which differed between endosperm and leaves. These data indicate that CRWNs are necessary to maintain the H3K27me3 landscape, with tissue-specific chromatin and transcriptional consequences.

Epigenetic Regulation During Plant Development and the Capacity for Epigenetic Memory.

The establishment, maintenance, and removal of epigenetic modifications provide an additional layer of regulation, beyond genetically encoded factors, by which plants can control developmental processes and adapt to the environment. Epigenetic inheritance, while historically referring to information not encoded in the DNA sequence that is inherited between generations, can also refer to epigenetic modifications that are maintained within an individual but are reset between generations. Both types of epigenetic inheritance occur in plants, and the functions and mechanisms distinguishing the two are of great interest to the field. Here, we discuss examples of epigenetic dynamics and maintenance during selected stages of growth and development and their functional consequences. Epigenetic states are also dynamic in response to stress, with consequences for transposable element regulation. How epigenetic resetting between generations occurs during normal development and in response to stress is an emerging area of research.

RNA Pol IV induces antagonistic parent-of-origin effects on Arabidopsis endosperm.

Gene expression in endosperm-a seed tissue that mediates transfer of maternal resources to offspring-is under complex epigenetic control. We show here that plant-specific RNA polymerase IV (Pol IV) mediates parental control of endosperm gene expression. Pol IV is required for the production of small interfering RNAs that typically direct DNA methylation. We compared small RNAs (sRNAs), DNA methylation, and mRNAs in Arabidopsis thaliana endosperm from heterozygotes produced by reciprocally crossing wild-type (WT) plants to Pol IV mutants. We find that maternally and paternally acting Pol IV induce distinct effects on endosperm. Loss of maternal or paternal Pol IV impacts sRNAs and DNA methylation at different genomic sites. Strikingly, maternally and paternally acting Pol IV have antagonistic impacts on gene expression at some loci, divergently promoting or repressing endosperm gene expression. Antagonistic parent-of-origin effects have only rarely been described and are consistent with a gene regulatory system evolving under parental conflict.

Somatic DNA demethylation generates tissue-specific methylation states and impacts flowering time.

Cytosine methylation is a reversible epigenetic modification of DNA. In plants, removal of cytosine methylation is accomplished by the four members of the DEMETER (DME) family of 5-methylcytosine DNA glycosylases, named DME, DEMETER-LIKE2 (DML2), DML3, and REPRESSOR OF SILENCING1 (ROS1) in Arabidopsis thaliana. Demethylation by DME is critical for seed development, preventing experiments to determine the function of the entire gene family in somatic tissues by mutant analysis. Here, we bypassed the reproductive defects of dme mutants to create somatic quadruple homozygous mutants of the entire DME family. dme; ros1; dml2; and dml3 (drdd) leaves exhibit hypermethylated regions compared with wild-type leaves and rdd triple mutants, indicating functional redundancy among all four demethylases. Targets of demethylation include regions co-targeted by RNA-directed DNA methylation and, surprisingly, CG gene body methylation, indicating dynamic methylation at these less-understood sites. Additionally, many tissue-specific methylation differences are absent in drdd, suggesting a role for active demethylation in generating divergent epigenetic states across wild-type tissues. Furthermore, drdd plants display an early flowering phenotype, which involves 5'-hypermethylation and transcriptional down-regulation of FLOWERING LOCUS C. Active DNA demethylation is therefore required for proper methylation across somatic tissues and defines the epigenetic landscape of intergenic and coding regions.

Maternal-filial transfer structures in endosperm: A nexus of nutritional dynamics and seed development.

Although the ultimate purpose of a seed is the successful establishment of the next generation, seed development involves more than embryo growth. In angiosperms, seed development requires the intimate coordination of three distinct entities - maternal tissue and two offspring, embryo and embryo-nourishing endosperm. Although seeds are cornerstones of many terrestrial ecosystems and human diets, we are only beginning to understand the interactions among seed tissues and the molecular processes and genes that determine them. Recent studies of gene expression and function in distantly related angiosperms, combined with over 100 years of embryological research, have repeatedly highlighted the endosperm associated with maternal-filial boundaries as a central point in seed developmental dynamics. In this review, we highlight evidence that links this zone with nutritional dynamics, developmental signaling, and imprinted gene expression. We suggest that the underappreciated diversity of this specialized endosperm across angiosperms deserves further study from developmental, molecular, and genetic perspectives.

Transcriptional and imprinting complexity in Arabidopsis seeds at single-nucleus resolution.

Seeds are a key life cycle stage for many plants. Seeds are also the basis of agriculture and the primary source of calories consumed by humans1. Here, we employ single-nucleus RNA-sequencing to generate a transcriptional atlas of developing Arabidopsis thaliana seeds, with a focus on endosperm. Endosperm, the primary site of gene imprinting in flowering plants, mediates the relationship between the maternal parent and the embryo2. We identify transcriptionally uncharacterized nuclei types in the chalazal endosperm, which interfaces with maternal tissue for nutrient unloading3,4. We demonstrate that the extent of parental bias of maternally expressed imprinted genes varies with cell-cycle phase, and that imprinting of paternally expressed imprinted genes is strongest in chalazal endosperm. Thus, imprinting is spatially and temporally heterogeneous. Increased paternal expression in the chalazal region suggests that parental conflict, which is proposed to drive imprinting evolution, is fiercest at the boundary between filial and maternal tissues.

Principles of Epigenetic Homeostasis Shared Between Flowering Plants and Mammals.

In diverse eukaryotes, epigenetic information such as DNA methylation is stably propagated over many cell divisions and generations, and can remain the same over thousands or millions of years. However, this stability is the product of dynamic processes that add and remove DNA methylation by specialized enzymatic pathways. The activities of these dynamic pathways must therefore be finely orchestrated in order to ensure that the DNA methylation landscape is maintained with high fidelity - a concept we term epigenetic homeostasis. In this review, we summarize recent insights into epigenetic homeostasis mechanisms in flowering plants and mammals, highlighting analogous mechanisms that have independently evolved to achieve the same goal of stabilizing the epigenetic landscape.

Water lily (Nymphaea thermarum) genome reveals variable genomic signatures of ancient vascular cambium losses.

For more than 225 million y, all seed plants were woody trees, shrubs, or vines. Shortly after the origin of angiosperms ∼140 million y ago (MYA), the Nymphaeales (water lilies) became one of the first lineages to deviate from their ancestral, woody habit by losing the vascular cambium, the meristematic population of cells that produces secondary xylem (wood) and phloem. Many of the genes and gene families that regulate differentiation of secondary tissues also regulate the differentiation of primary xylem and phloem, which are produced by apical meristems and retained in nearly all seed plants. Here, we sequenced and assembled a draft genome of the water lily Nymphaea thermarum, an emerging system for the study of early flowering plant evolution, and compared it to genomes from other cambium-bearing and cambium-less lineages (e.g., monocots and Nelumbo). This revealed lineage-specific patterns of gene loss and divergence. Nymphaea is characterized by a significant contraction of the HD-ZIP III transcription factors, specifically loss of REVOLUTA, which influences cambial activity in other angiosperms. We also found the Nymphaea and monocot copies of cambium-associated CLE signaling peptides display unique substitutions at otherwise highly conserved amino acids. Nelumbo displays no obvious divergence in cambium-associated genes. The divergent genomic signatures of convergent loss of vascular cambium reveals that even pleiotropic genes can exhibit unique divergence patterns in association with independent events of trait loss. Our results shed light on the evolution of herbaceousness-one of the key biological innovations associated with the earliest phases of angiosperm evolution.

Identification and Comparison of Imprinted Genes Across Plant Species.

Genomic imprinting is a phenomenon that occurs in flowering plants and mammals, whereby a gene is expressed in a parent-of-origin-specific manner. Although imprinting has now been examined genome-wide in a number of species using RNA-seq, the analyses used to assess imprinting vary between studies, making consistent comparisons between species difficult. Here we present a simple, easy-to-use bioinformatic pipeline for imprinting analyses suitable for any tissue, including plant endosperm. All relevant scripts can be downloaded. As an illustrative example, we reanalyze published data from A. thaliana and Z. mays endosperm using the pipeline and then demonstrate how to use the results to assess the conservation of imprinting between these species. We also introduce the Plant Imprinting Database, a repository for published imprinting datasets in plants that can be used to view, compare, and download data.

Paternally Acting Canonical RNA-Directed DNA Methylation Pathway Genes Sensitize Arabidopsis Endosperm to Paternal Genome Dosage.

Seed development is sensitive to parental dosage, with excess maternal or paternal genomes creating reciprocal phenotypes. Paternal genomic excess frequently results in extensive endosperm proliferation without cellularization and seed abortion. We previously showed that loss of the RNA polymerase IV gene NUCLEAR RNA POLYMERASE D1 (NRPD1) in tetraploid fathers represses seed abortion in paternal excess crosses. Here, we show genetically that RNA-directed DNA methylation (RdDM) pathway activity in the paternal parent is sufficient to determine the viability of paternal excess Arabidopsis (Arabidopsis thaliana) seeds. We compared transcriptomes, DNA methylation, and small RNAs from the endosperm of seeds from balanced crosses (diploid × diploid) and lethal (diploid × tetraploid) and viable paternal excess crosses (diploid × tetraploid nrpd1). Endosperms from both lethal and viable paternal excess seeds share widespread transcriptional and DNA methylation changes at genes and transposable elements. Interploidy seed abortion is thus unlikely to be caused by transposable elements or imprinted gene misregulation, and its repression by the loss of paternal RdDM is associated with only modest gene expression changes. Finally, using allele-specific transcription data, we present evidence for a transcriptional buffering system that increases the expression of maternal alleles and represses paternal alleles in response to excess paternal genomic dosage. These findings prompt reconsideration of models for dosage sensitivity in endosperm.

Epigenetic dynamics during flowering plant reproduction: evidence for reprogramming?

Over the last 10 yr there have been major advances in documenting and understanding dynamic changes to DNA methylation, small RNAs, chromatin modifications and chromatin structure that accompany reproductive development in flowering plants, from germline specification to seed maturation. Here I highlight recent advances in the field, mostly made possible by microscopic analysis of epigenetic states or by the ability to isolate specific cell types or tissues and apply omics approaches. I consider in which contexts there is potentially reprogramming vs maintenance or reinforcement of epigenetic states.

Low-input chromatin profiling in Arabidopsis endosperm using CUT&RUN.

KEY MESSAGE: Application of a low-input chromatin profiling method, CUT&RUN, to FACS-purified Arabidopsis endosperm nuclei generates parental-specific genome-wide H3K27me3 landscapes with high sensitivity, specificity and reproducibility. Endosperm is an essential seed tissue with a unique epigenetic landscape. During endosperm development, differential epigenetic regulation of the maternal and paternal genomes plays important roles in regulating gene expression, especially at imprinted genes. In Arabidopsis, profiling the epigenetic landscape of endosperm on a genome-wide scale is challenging due to its small size, mode of development and close association with maternal tissue. Here, we applied a low-input chromatin profiling method, CUT&RUN (cleavage under targets and release using nuclease), to profile parental-specific chromatin modifications using limited numbers of Arabidopsis endosperm nuclei. We demonstrate that CUT&RUN generates genome-wide H3K27me3 landscapes with high sensitivity, specificity and reproducibility using around 20,000 endosperm nuclei purified by flow cytometry and fluorescence-activated cell sorting. H3K27me3 peaks identified by CUT&RUN and previous ChIP (chromatin immunoprecipitation) approaches were largely overlapping, with some distinctions in heterochromatin. The versatility and simplicity of CUT&RUN make it a viable alternative to ChIP, which requires greater amounts of starting material, and will enable further study of tissue- or cell-type-specific epigenomes in Arabidopsis and other plant species.

Ten things you should know about transposable elements.

Transposable elements (TEs) are major components of eukaryotic genomes. However, the extent of their impact on genome evolution, function, and disease remain a matter of intense interrogation. The rise of genomics and large-scale functional assays has shed new light on the multi-faceted activities of TEs and implies that they should no longer be marginalized. Here, we introduce the fundamental properties of TEs and their complex interactions with their cellular environment, which are crucial to understanding their impact and manifold consequences for organismal biology. While we draw examples primarily from mammalian systems, the core concepts outlined here are relevant to a broad range of organisms.

A variably imprinted epiallele impacts seed development.

The contribution of epigenetic variation to phenotypic variation is unclear. Imprinted genes, because of their strong association with epigenetic modifications, represent an opportunity for the discovery of such phenomena. In mammals and flowering plants, a subset of genes are expressed from only one parental allele in a process called gene imprinting. Imprinting is associated with differential DNA methylation and chromatin modifications between parental alleles. In flowering plants imprinting occurs in a seed tissue - endosperm. Proper endosperm development is essential for the production of viable seeds. We previously showed that in Arabidopsis thaliana intraspecific imprinting variation is correlated with naturally occurring DNA methylation polymorphisms. Here, we investigated the mechanisms and function of allele-specific imprinting of the class IV homeodomain leucine zipper (HD-ZIP) transcription factor HDG3. In imprinted strains, HDG3 is expressed primarily from the methylated paternally inherited allele. We manipulated the methylation state of endogenous HDG3 in a non-imprinted strain and demonstrated that methylation of a proximal transposable element is sufficient to promote HDG3 expression and imprinting. Gain of HDG3 imprinting was associated with earlier endosperm cellularization and changes in seed weight. These results indicate that epigenetic variation alone is sufficient to explain imprinting variation and demonstrate that epialleles can underlie variation in seed development phenotypes.

A Small RNA Pathway Mediates Allelic Dosage in Endosperm.

Balance between maternal and paternal genomes within the triploid endosperm is necessary for normal seed development. The majority of endosperm genes are expressed in a 2:1 maternal:paternal ratio, reflecting genomic DNA content. Here, we find that the 2:1 transcriptional ratio is, unexpectedly, actively regulated. In A. thaliana and A. lyrata, endosperm 24-nt small RNAs are reduced in transposable elements and enriched in genes compared with the embryo. We find an inverse relationship between the parent of origin of sRNAs and mRNAs, with genes more likely to be associated with maternally than paternally biased sRNAs. Disruption of the Pol IV sRNA pathway causes a shift toward maternal allele mRNA expression for many genes. Furthermore, paternal inheritance of an RNA Pol IV mutation is sufficient to rescue seed abortion caused by excess paternal genome dosage. Thus, RNA Pol IV mediates the transcriptional balance between maternally and paternally inherited genomes in endosperm.

Stable transgenerational epigenetic inheritance requires a DNA methylation-sensing circuit.

Epigenetic states are stably propagated in eukaryotes. In plants, DNA methylation patterns are faithfully inherited over many generations but it is unknown how the dynamic activities of cytosine DNA methyltransferases and 5-methylcytosine DNA glycosylases interact to maintain epigenetic homeostasis. Here we show that a methylation-sensing gene regulatory circuit centered on a 5-methylcytosine DNA glycosylase gene is required for long-term epigenetic fidelity in Arabidopsis. Disrupting this circuit causes widespread methylation losses and abnormal phenotypes that progressively worsen over generations. In heterochromatin, these losses are counteracted such that methylation returns to a normal level over four generations. However, thousands of loci in euchromatin progressively lose DNA methylation between generations and remain unmethylated. We conclude that an actively maintained equilibrium between methylation and demethylation activities is required to ensure long-term stable inheritance of epigenetic information.

Molecular movement in the Arabidopsis thaliana female gametophyte.

KEY MESSAGE: Size limits on molecular movement among female gametes. Cellular decisions can be influenced by information communicated from neighboring cells. Communication can occur via signaling or through the direct transfer of molecules. Movement of RNAs and proteins has frequently been observed among symplastically connected plant cells. In flowering plants, the female gametes, the egg cell and central cell, are closely apposed within the female gametophyte. Here we investigated the ability of fluorescently labeled dyes and small RNAs to move from the Arabidopsis thaliana central cell to the egg apparatus following microinjection. These results define a size limit of at least 20 kDa for symplastic movement between the two gametes, somewhat larger than that previously observed in Torenia fournieri. Our results indicate that symplastic connectivity in Arabidopsis thaliana changes after fertilization and suggest that prior to fertilization mechanisms are in place to facilitate small RNA movement from the central cell to the egg cell and synergids.

Proximal methylation features associated with nonrandom changes in gene body methylation.

BACKGROUND: Gene body methylation at CG dinucleotides is a widely conserved feature of methylated genomes but remains poorly understood. The Arabidopsis thaliana strain Cvi has depleted gene body methylation relative to the reference strain Col. Here, we leverage this natural epigenetic difference to investigate gene body methylation stability. RESULTS: Recombinant inbred lines derived from Col and Cvi were used to examine the transmission of distinct gene body methylation states. The vast majority of genic CG methylation patterns are faithfully transmitted over nine generations according to parental genotype, with only 1-4% of CGs either losing or gaining methylation relative to the parent. Genic CGs that fail to maintain the parental methylation state are shared among independent lines, suggesting that these are not random occurrences. We use a logistic regression framework to identify features that best predict sites that fail to maintain parental methylation state. Intermediate levels of CG methylation around a dynamic CG site and high methylation variability across many A. thaliana strains at that site are the strongest predictors. These data suggest that the dynamic CGs we identify are not specific to the Col-Cvi recombinant inbred lines but have an epigenetic state that is inherently less stable within the A. thaliana species. Extending this, variably methylated genic CGs in maize and Brachypodium distachyon are also associated with intermediate local CG methylation. CONCLUSIONS: These results provide new insights into the features determining the inheritance of gene body methylation and demonstrate that two different methylation equilibria can be maintained within single individuals.

DNA methylation and imprinting in plants: machinery and mechanisms.

Imprinting is an epigenetic phenomenon in which genes are expressed selectively from either the maternal or paternal alleles. In plants, imprinted gene expression is found in a tissue called the endosperm. Imprinting is often set by a unique epigenomic configuration in which the maternal chromosomes are less DNA methylated than their paternal counterparts. In this review, we synthesize studies that paint a detailed molecular portrait of the distinctive endosperm methylome. We will also discuss the molecular machinery that shapes and modifies this methylome, and the role of DNA methylation in imprinting.