DNA methylation signatures of duplicate gene evolution in angiosperms.

Gene duplication is a source of evolutionary novelty. DNA methylation may play a role in the evolution of duplicate genes (paralogs) through its association with gene expression. While this relationship has been examined to varying extents in a few individual species, the generalizability of these results at either a broad phylogenetic scale with species of differing duplication histories or across a population remains unknown. We applied a comparative epigenomic approach to 43 angiosperm species across the phylogeny and a population of 928 Arabidopsis (Arabidopsis thaliana) accessions, examining the association of DNA methylation with paralog evolution. Genic DNA methylation was differentially associated with duplication type, the age of duplication, sequence evolution, and gene expression. Whole-genome duplicates were typically enriched for CG-only gene body methylated or unmethylated genes, while single-gene duplications were typically enriched for non-CG methylated or unmethylated genes. Non-CG methylation, in particular, was a characteristic of more recent single-gene duplicates. Core angiosperm gene families were differentiated into those which preferentially retain paralogs and "duplication-resistant" families, which convergently reverted to singletons following duplication. Duplication-resistant families that still have paralogous copies were, uncharacteristically for core angiosperm genes, enriched for non-CG methylation. Non-CG methylated paralogs had higher rates of sequence evolution, higher frequency of presence-absence variation, and more limited expression. This suggests that silencing by non-CG methylation may be important to maintaining dosage following duplication and be a precursor to fractionation. Our results indicate that genic methylation marks differing evolutionary trajectories and fates between paralogous genes and have a role in maintaining dosage following duplication.

pENCODE: a plant encyclopedia of DNA elements.

ENCODE projects exist for many eukaryotes, including humans, but as of yet no defined project exists for plants. A plant ENCODE would be invaluable to the research community and could be more readily produced than its metazoan equivalents by capitalizing on the preexisting infrastructure provided from similar projects. Collecting and normalizing plant epigenomic data for a range of species will facilitate hypothesis generation, cross-species comparisons, annotation of genomes, and an understanding of epigenomic functions throughout plant evolution. Here, we discuss the need for such a project, outline the challenges it faces, and suggest ways forward to build a plant ENCODE.

Prenatal testosterone triggers long-term behavioral changes in male zebra finches: unravelling the neurogenomic mechanisms.

BACKGROUND: Maternal hormones, like testosterone, can strongly influence developing offspring, even generating long-term organizational effects on adult behavior; yet, the mechanisms facilitating these effects are still unclear. Here, we experimentally elevated prenatal testosterone in the eggs of zebra finches (Taeniopygia guttata) and measured male aggression in adulthood along with patterns of neural gene expression (RNA-seq) and DNA methylation (MethylC-Seq) in two socially relevant brain regions (hypothalamus and nucleus taenia of the amygdala). We used enrichment analyses and protein-protein interaction networks to find candidate processes and hub genes potentially affected by the treatment. We additionally identified differentially expressed genes that contained differentially methylated regions. RESULTS: We found that males from testosterone-injected eggs displayed more aggressive behaviors compared to males from control eggs. Hundreds of genes were differentially expressed, particularly in the hypothalamus, including potential aggression-related hub genes (e.g., brain derived neurotrophic factor). There were also enriched processes with well-established links to aggressive phenotypes (e.g., somatostatin and glutamate signaling). Furthermore, several highly connected genes identified in protein-protein interaction networks also showed differential methylation, including adenylate cyclase 2 and proprotein convertase 2. CONCLUSIONS: These results highlight genes and processes that may play an important role in mediating the effects of prenatal testosterone on long-term phenotypic outcomes, thereby providing insights into the molecular mechanisms that facilitate hormone-mediated maternal effects.

Replaying the evolutionary tape to investigate subgenome dominance in allopolyploid Brassica napus.

Allopolyploidisation merges evolutionarily distinct parental genomes (subgenomes) into a single nucleus. A frequent observation is that one subgenome is 'dominant' over the other subgenome, often being more highly expressed. Here, we 'replayed the evolutionary tape' with six isogenic resynthesised Brassica napus allopolyploid lines and investigated subgenome dominance patterns over the first 10 generations postpolyploidisation. We found that the same subgenome was consistently more dominantly expressed in all lines and generations and that >70% of biased gene pairs showed the same dominance patterns across all lines and an in silico hybrid of the parents. Gene network analyses indicated an enrichment for network interactions and several biological functions for the Brassica oleracea subgenome biased pairs, but no enrichment was identified for Brassica rapa subgenome biased pairs. Furthermore, DNA methylation differences between subgenomes mirrored the observed gene expression bias towards the dominant subgenome in all lines and generations. Many of these differences in gene expression and methylation were also found when comparing the progenitor genomes, suggesting that subgenome dominance is partly related to parental genome differences rather than just a byproduct of allopolyploidisation. These findings demonstrate that 'replaying the evolutionary tape' in an allopolyploid results in largely repeatable and predictable subgenome expression dominance patterns.

Epigenetics and epigenomics: underlying mechanisms, relevance, and implications in crop improvement.

Epigenetics is defined as changes in gene expression that are not associated with changes in DNA sequence but due to the result of methylation of DNA and post-translational modifications to the histones. These epigenetic modifications are known to regulate gene expression by bringing changes in the chromatin state, which underlies plant development and shapes phenotypic plasticity in responses to the environment and internal cues. This review articulates the role of histone modifications and DNA methylation in modulating biotic and abiotic stresses, as well as crop improvement. It also highlights the possibility of engineering epigenomes and epigenome-based predictive models for improving agronomic traits.

On the origin and evolutionary consequences of gene body DNA methylation.

In plants, CG DNA methylation is prevalent in the transcribed regions of many constitutively expressed genes (gene body methylation; gbM), but the origin and function of gbM remain unknown. Here we report the discovery that Eutrema salsugineum has lost gbM from its genome, to our knowledge the first instance for an angiosperm. Of all known DNA methyltransferases, only CHROMOMETHYLASE 3 (CMT3) is missing from E. salsugineum Identification of an additional angiosperm, Conringia planisiliqua, which independently lost CMT3 and gbM, supports that CMT3 is required for the establishment of gbM. Detailed analyses of gene expression, the histone variant H2A.Z, and various histone modifications in E. salsugineum and in Arabidopsis thaliana epigenetic recombinant inbred lines found no evidence in support of any role for gbM in regulating transcription or affecting the composition and modification of chromatin over evolutionary timescales.

Hypermethylation and small RNA expression are associated with increased age in almond (Prunus dulcis [Mill.] D.A. Webb) accessions.

The focus of this study is to profile changes in DNA methylation and small RNA expression occurring with increased age in almond breeding germplasm to identify possible biomarkers of age that can be used to assess the potential of individuals to develop aging-related disorders. To profile DNA methylation in almond germplasm, 70 methylomes were generated from almond individuals representing three age cohorts (11, 7, and 2 years old) using an enzymatic methyl-seq approach followed by analysis to call differentially methylated regions (DMRs) within these cohorts. Small RNA (sRNA) expression was profiled in three breeding selections, each from two age cohorts (1 and 6 years old), using sRNA-Seq followed by differential expression analysis. Weighted chromosome-level methylation analysis reveals hypermethylation in 11-year-old almond breeding selections when compared to 2-year-old selections in the CG and CHH contexts. Seventeen consensus DMRs were identified in all age contrasts. sRNA expression differed significantly between the two age cohorts tested, with significantly decreased expression in sRNAs in the 6-year-old selections compared to the 1-year-old. Almond shows a pattern of hypermethylation and decreased sRNA expression with increased age. Identified DMRs and differentially expressed sRNAs could function as putative biomarkers of age following validation in additional age groups.

Transcriptional profiling of the Arabidopsis abscission mutant hae hsl2 by RNA-Seq.

BACKGROUND: Abscission is a mechanism by which plants shed entire organs in response to both developmental and environmental signals. Arabidopsis thaliana, in which only the floral organs abscise, has been used extensively to study the genetic, molecular and cellular processes controlling abscission. Abscission in Arabidopsis requires two genes that encode functionally redundant receptor-like protein kinases, HAESA (HAE) and HAESA-LIKE 2 (HSL2). Double hae hsl2 mutant plants fail to abscise their floral organs at any stage of floral development and maturation. RESULTS: Using RNA-Seq, we compare the transcriptomes of wild-type and hae hsl2 stage 15 flowers, using the floral receptacle which is enriched for abscission zone cells. 2034 genes were differentially expressed with a False Discovery Rate adjusted p < 0.05, of which 349 had two fold or greater change in expression. Differentially expressed genes were enriched for hydrolytic, cell wall modifying, and defense related genes. Testing several of the differentially expressed genes in INFLORESCENCE DEFICIENT IN ABSCISSION (ida) mutants shows that many of the same genes are co-regulated by IDA and HAE HSL2 and support the role of IDA in the HAE and HSL2 signaling pathway. Comparison to microarray data from stamen abscission zones show distinct patterns of expression of genes that are dependent on HAE HSL2 and reveal HAE HSL2- independent pathways. CONCLUSION: HAE HSL2-dependent and HAE HSL2-independent changes in genes expression are required for abscission. HAE and HSL2 affect the expression of cell wall modifying and defense related genes necessary for abscission. The HAE HSL2-independent genes also appear to have roles in abscission and additionally are involved in processes such as hormonal signaling, senescence and callose deposition.

Letting go is never easy: abscission and receptor-like protein kinases.

Abscission is the process by which plants discard organs in response to environmental cues/stressors, or as part of their normal development. Abscission has been studied throughout the history of the plant sciences and in numerous species. Although long studied at the anatomical and physiological levels, abscission has only been elucidated at the molecular and genetic levels within the last two decades, primarily with the use of the model plant Arabidopsis thaliana. This has led to the discovery of numerous genes involved at all steps of abscission, including key pathways involving receptor-like protein kinases (RLKs). This review covers the current knowledge of abscission research, highlighting the role of RLKs. [Figure: see text] John C. Walker (Corresponding author).

Maternal dominance contributes to subgenome differentiation in allopolyploid fishes.

Teleost fishes, which are the largest and most diverse group of living vertebrates, have a rich history of ancient and recent polyploidy. Previous studies of allotetraploid common carp and goldfish (cyprinids) reported a dominant subgenome, which is more expressed and exhibits biased gene retention. However, the underlying mechanisms contributing to observed 'subgenome dominance' remains poorly understood. Here we report high-quality genomes of twenty-one cyprinids to investigate the origin and subsequent subgenome evolution patterns following three independent allopolyploidy events. We identify the closest extant relatives of the diploid progenitor species, investigate genetic and epigenetic differences among subgenomes, and conclude that observed subgenome dominance patterns are likely due to a combination of maternal dominance and transposable element densities in each polyploid. These findings provide an important foundation to understanding subgenome dominance patterns observed in teleost fishes, and ultimately the role of polyploidy in contributing to evolutionary innovations.

DVL genes play a role in the coordination of socket cell recruitment and differentiation.

Specialized plant cells arise from undifferentiated cells through a series of developmental steps. The decision to enter into a certain differentiation pathway depends in many cases on signals from neighbouring cells. The ability of cells to engage in short-range intercellular communication permits the coordination of cell actions necessary in many developmental processes. Overexpression of genes from the DEVIL/ROTUNDIFOLIA (DVL/ROT) family results in severe developmental alterations, but very little is known about their mechanism of action. This work presents evidence that suggests a role for these genes in local signalling, specifically in the coordination of socket cell recruitment and differentiation. Overexpression of different DVL genes results in protuberances at the base of the trichomes surrounded by several rows of elongated epidermal cells, morphologically similar to socket cells. Localized overexpression of DVL4 in trichomes and socket cells during early developmental stages activates expression of socket cell markers in additional cells, farther away from the trichome. The same phenomenon is observed in an activation tagged line of DVL1, which also shows an increase in the number of socket cells in contact with the trichome. The roles of individual DVL genes have been difficult to discover since their overexpression phenotypes are quite similar. In gl1 leaves that lack trichomes and socket cells DVL1 expression shows a 69% reduction, suggesting that this gene could be involved in the coordination of socket cell development in wild-type plants.

Regulatory dynamics distinguishing desiccation tolerance strategies within resurrection grasses.

Desiccation tolerance has evolved recurrently in grasses using two unique strategies of either protecting or dismantling the photosynthetic apparatus to minimize photooxidative damage under life without water (anhydrobiosis). Here, we surveyed chromatin architecture and gene expression during desiccation in two closely related grasses with distinguishing desiccation tolerance strategies to identify regulatory dynamics underlying these unique adaptations. In both grasses, we observed a strong association between nearby chromatin accessibility and gene expression in desiccated tissues compared to well-watered, reflecting an unusual chromatin stability under anhydrobiosis. Integration of chromatin accessibility (ATACseq) and expression data (RNAseq) revealed a core desiccation response across these two grasses. This includes many genes with binding sites for the core seed development transcription factor ABI5, supporting the long-standing hypothesis that vegetative desiccation tolerance evolved from rewiring seed pathways. Oropetium thomaeum has a unique set of desiccation induced genes and regulatory elements associated with photoprotection, pigment biosynthesis, and response to high light, reflecting its adaptation of protecting the photosynthetic apparatus under desiccation (homoiochlorophyly). By contrast, Eragrostis nindensis has unique accessible and expressed genes related to chlorophyll catabolism, scavenging of amino acids, and hypoxia, highlighting its poikilochlorophyllous adaptations of dismantling the photosynthetic apparatus and degrading chlorophyll under desiccation. Together, our results highlight the complex regulatory and expression dynamics underlying desiccation tolerance in grasses.

Intertwined evolution of plant epigenomes and genomes.

DNA methylation is found across eukaryotes; however, plants have evolved patterns and pathways of DNA methylation that are distinct from animals and fungi. DNA methylation shapes the evolution of genomes through its direct roles in transposon silencing, gene expression, genome stability, and its impact on mutation rates. In return the diversity of DNA methylation across species is shaped by genome sequence evolution. Extensive diversification of key DNA methylation pathways has continued in plants through gene duplication and loss. Meanwhile, frequent movement of transposons has altered local DNA methylation patterns and the genes affected. Only recently has the diversity and evolutionary history of plant DNA methylation become evident with the availability of increasing genomic and epigenomic data. However, much remains unresolved regarding the evolutionary forces that have shaped the dynamics of the complex and intertwined history of plant genome and epigenome evolution.

The Molecular Basis of Kale Domestication: Transcriptional Profiling of Developing Leaves Provides New Insights Into the Evolution of a Brassica oleracea Vegetative Morphotype.

Morphotypes of Brassica oleracea are the result of a dynamic interaction between genes that regulate the transition between vegetative and reproductive stages and those that regulate leaf morphology and plant architecture. In kales, ornate leaves, extended vegetative phase, and nutritional quality are some of the characters potentially selected by humans during domestication. We used a combination of developmental studies and transcriptomics to understand the vegetative domestication syndrome of kale. To identify candidate genes that are responsible for the evolution of domestic kale, we searched for transcriptome-wide differences among three vegetative B. oleracea morphotypes. RNA-seq experiments were used to understand the global pattern of expressed genes during a mixture of stages at one time in kale, cabbage, and the rapid cycling kale line TO1000. We identified gene expression patterns that differ among morphotypes and estimate the contribution of morphotype-specific gene expression that sets kale apart (3958 differentially expressed genes). Differentially expressed genes that regulate the vegetative to reproductive transition were abundant in all morphotypes. Genes involved in leaf morphology, plant architecture, defense, and nutrition were differentially expressed in kale. This allowed us to identify a set of candidate genes we suggest may be important in the kale domestication syndrome. Understanding candidate genes responsible for kale domestication is of importance to ultimately improve Cole crop production.

Establishment, maintenance, and biological roles of non-CG methylation in plants.

Cytosine DNA methylation is prevalent throughout eukaryotes and prokaryotes. While most commonly thought of as being localized to dinucleotide CpG sites, non-CG sites can also be modified. Such non-CG methylation is widespread in plants, occurring at trinucleotide CHG and CHH (H = A, T, or C) sequence contexts. The prevalence of non-CG methylation in plants is due to the plant-specific CHROMOMETHYLASE (CMT) and RNA-directed DNA Methylation (RdDM) pathways. These pathways have evolved through multiple rounds of gene duplication and gene loss, generating epigenomic variation both within and between species. They regulate both transposable elements and genes, ensure genome integrity, and ultimately influence development and environmental responses. In these capacities, non-CG methylation influence and shape plant genomes.

Single-molecule sequencing and optical mapping yields an improved genome of woodland strawberry (Fragaria vesca) with chromosome-scale contiguity.

Background: Although draft genomes are available for most agronomically important plant species, the majority are incomplete, highly fragmented, and often riddled with assembly and scaffolding errors. These assembly issues hinder advances in tool development for functional genomics and systems biology. Findings: Here we utilized a robust, cost-effective approach to produce high-quality reference genomes. We report a near-complete genome of diploid woodland strawberry (Fragaria vesca) using single-molecule real-time sequencing from Pacific Biosciences (PacBio). This assembly has a contig N50 length of ∼7.9 million base pairs (Mb), representing a ∼300-fold improvement of the previous version. The vast majority (>99.8%) of the assembly was anchored to 7 pseudomolecules using 2 sets of optical maps from Bionano Genomics. We obtained ∼24.96 Mb of sequence not present in the previous version of the F. vesca genome and produced an improved annotation that includes 1496 new genes. Comparative syntenic analyses uncovered numerous, large-scale scaffolding errors present in each chromosome in the previously published version of the F. vesca genome. Conclusions: Our results highlight the need to improve existing short-read based reference genomes. Furthermore, we demonstrate how genome quality impacts commonly used analyses for addressing both fundamental and applied biological questions.

Putting DNA methylation in context: from genomes to gene expression in plants.

Plant DNA methylation is its own language, interpreted by the cell to maintain silencing of transposons, facilitate chromatin structure, and to ensure proper expression of some genes. Just as in any language, context is important. Rather than being a simple "on-off switch", DNA methylation has a range of "meanings" dependent upon the underlying sequence and its location in the genome. Differences in the sequence context of individual sites are established, maintained, and interpreted by differing molecular pathways. Varying patterns of methylation within genes and surrounding sequences are associated with a continuous range of expression differences, from silencing to constitutive expression. These often-subtle differences have been pieced together from years of effort, but have taken off with the advent of methods for assessing methylation across entire genomes. Recognizing these patterns and identifying underlying causes is essential for understanding the function of DNA methylation and its systems-wide contribution to a range of processes in plant genomes. This article is part of a Special Issue entitled: Plant Gene Regulatory Mechanisms and Networks, edited by Dr. Erich Grotewold and Dr. Nathan Springer.

The evolution of CHROMOMETHYLASES and gene body DNA methylation in plants.

BACKGROUND: The evolution of gene body methylation (gbM), its origins, and its functional consequences are poorly understood. By pairing the largest collection of transcriptomes (>1000) and methylomes (77) across Viridiplantae, we provide novel insights into the evolution of gbM and its relationship to CHROMOMETHYLASE (CMT) proteins. RESULTS: CMTs are evolutionary conserved DNA methyltransferases in Viridiplantae. Duplication events gave rise to what are now referred to as CMT1, 2 and 3. Independent losses of CMT1, 2, and 3 in eudicots, CMT2 and ZMET in monocots and monocots/commelinids, variation in copy number, and non-neutral evolution suggests overlapping or fluid functional evolution of this gene family. DNA methylation within genes is widespread and is found in all major taxonomic groups of Viridiplantae investigated. Genes enriched with methylated CGs (mCG) were also identified in species sister to angiosperms. The proportion of genes and DNA methylation patterns associated with gbM are restricted to angiosperms with a functional CMT3 or ortholog. However, mCG-enriched genes in the gymnosperm Pinus taeda shared some similarities with gbM genes in Amborella trichopoda. Additionally, gymnosperms and ferns share a CMT homolog closely related to CMT2 and 3. Hence, the dependency of gbM on a CMT most likely extends to all angiosperms and possibly gymnosperms and ferns. CONCLUSIONS: The resulting gene family phylogeny of CMT transcripts from the most diverse sampling of plants to date redefines our understanding of CMT evolution and its evolutionary consequences on DNA methylation. Future, functional tests of homologous and paralogous CMTs will uncover novel roles and consequences to the epigenome.

A Comparative Analysis of 5-Azacytidine- and Zebularine-Induced DNA Demethylation.

The nonmethylable cytosine analogs, 5-azacytidine and zebularine, are widely used to inhibit DNA methyltransferase activity and reduce genomic DNA methylation. In this study, whole-genome bisulfite sequencing is used to construct maps of DNA methylation with single base pair resolution in Arabidopsis thaliana seedlings treated with each demethylating agent. We find that both inhibitor treatments result in nearly indistinguishable patterns of genome-wide DNA methylation and that 5-azacytidine had a slightly greater demethylating effect at higher concentrations across the genome. Transcriptome analyses revealed a substantial number of upregulated genes, with an overrepresentation of transposable element genes, in particular CACTA-like elements. This demonstrates that chemical demethylating agents have a disproportionately large effect on loci that are otherwise silenced by DNA methylation.

Widespread natural variation of DNA methylation within angiosperms.

BACKGROUND: DNA methylation is an important feature of plant epigenomes, involved in the formation of heterochromatin and affecting gene expression. Extensive variation of DNA methylation patterns within a species has been uncovered from studies of natural variation. However, the extent to which DNA methylation varies between flowering plant species is still unclear. To understand the variation in genomic patterning of DNA methylation across flowering plant species, we compared single base resolution DNA methylomes of 34 diverse angiosperm species. RESULTS: By analyzing whole-genome bisulfite sequencing data in a phylogenetic context, it becomes clear that there is extensive variation throughout angiosperms in gene body DNA methylation, euchromatic silencing of transposons and repeats, as well as silencing of heterochromatic transposons. The Brassicaceae have reduced CHG methylation levels and also reduced or loss of CG gene body methylation. The Poaceae are characterized by a lack or reduction of heterochromatic CHH methylation and enrichment of CHH methylation in genic regions. Furthermore, low levels of CHH methylation are observed in a number of species, especially in clonally propagated species. CONCLUSIONS: These results reveal the extent of variation in DNA methylation in angiosperms and show that DNA methylation patterns are broadly a reflection of the evolutionary and life histories of plant species.