Dynamic DNA methylation turnover in gene bodies is associated with enhanced gene expression plasticity in plants.

BACKGROUND: In several eukaryotes, DNA methylation occurs within the coding regions of many genes, termed gene body methylation (GbM). Whereas the role of DNA methylation on the silencing of transposons and repetitive DNA is well understood, gene body methylation is not associated with transcriptional repression, and its biological importance remains unclear. RESULTS: We report a newly discovered type of GbM in plants, which is under constitutive addition and removal by dynamic methylation modifiers in all cells, including the germline. Methylation at Dynamic GbM genes is removed by the DRDD demethylation pathway and added by an unknown source of de novo methylation, most likely the maintenance methyltransferase MET1. We show that the Dynamic GbM state is present at homologous genes across divergent lineages spanning over 100 million years, indicating evolutionary conservation. We demonstrate that Dynamic GbM is tightly associated with the presence of a promoter or regulatory chromatin state within the gene body, in contrast to other gene body methylated genes. We find Dynamic GbM is associated with enhanced gene expression plasticity across development and diverse physiological conditions, whereas stably methylated GbM genes exhibit reduced plasticity. Dynamic GbM genes exhibit reduced dynamic range in drdd mutants, indicating a causal link between DNA demethylation and enhanced gene expression plasticity. CONCLUSIONS: We propose a new model for GbM in regulating gene expression plasticity, including a novel type of GbM in which increased gene expression plasticity is associated with the activity of DNA methylation writers and erasers and the enrichment of a regulatory chromatin state.

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.

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.

Ancient duons may underpin spatial patterning of gene expression in C4 leaves.

If the highly efficient C4 photosynthesis pathway could be transferred to crops with the C3 pathway there could be yield gains of up to 50%. It has been proposed that the multiple metabolic and developmental modifications associated with C4 photosynthesis are underpinned by relatively few master regulators that have allowed the evolution of C4 photosynthesis more than 60 times in flowering plants. Here we identify a component of one such regulator that consists of a pair of cis-elements located in coding sequence of multiple genes that are preferentially expressed in bundle sheath cells of C4 leaves. These motifs represent duons as they play a dual role in coding for amino acids as well as controlling the spatial patterning of gene expression associated with the C4 leaf. They act to repress transcription of C4 photosynthesis genes in mesophyll cells. These duons are also present in the C3 model Arabidopsis thaliana, and, in fact, are conserved in all land plants and even some algae that use C3 photosynthesis. C4 photosynthesis therefore appears to have coopted an ancient regulatory code to generate the spatial patterning of gene expression that is a hallmark of C4 photosynthesis. This intragenic transcriptional regulatory sequence could be exploited in the engineering of efficient photosynthesis of crops.

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.

Evolutionary Inference across Eukaryotes Identifies Specific Pressures Favoring Mitochondrial Gene Retention.

Since their endosymbiotic origin, mitochondria have lost most of their genes. Although many selective mechanisms underlying the evolution of mitochondrial genomes have been proposed, a data-driven exploration of these hypotheses is lacking, and a quantitatively supported consensus remains absent. We developed HyperTraPS, a methodology coupling stochastic modeling with Bayesian inference, to identify the ordering of evolutionary events and suggest their causes. Using 2015 complete mitochondrial genomes, we inferred evolutionary trajectories of mtDNA gene loss across the eukaryotic tree of life. We find that proteins comprising the structural cores of the electron transport chain are preferentially encoded within mitochondrial genomes across eukaryotes. A combination of high GC content and high protein hydrophobicity is required to explain patterns of mtDNA gene retention; a model that accounts for these selective pressures can also predict the success of artificial gene transfer experiments in vivo. This work provides a general method for data-driven inference of the ordering of evolutionary and progressive events, here identifying the distinct features shaping mitochondrial genomes of present-day species.

An Untranslated cis-Element Regulates the Accumulation of Multiple C4 Enzymes in Gynandropsis gynandra Mesophyll Cells.

C4 photosynthesis is a complex phenotype that allows more efficient carbon capture than the ancestral C3 pathway. In leaves of C4 species, hundreds of transcripts increase in abundance compared with C3 relatives and become restricted to mesophyll (M) or bundle sheath (BS) cells. However, no mechanism has been reported that regulates the compartmentation of multiple enzymes in M or BS cells. We examined mechanisms regulating CARBONIC ANHYDRASE4 (CA4) in C4 Gynandropsis gynandra. Increased abundance is directed by both the promoter region and introns of the G. gynandra gene. A nine-nucleotide motif located in the 5' untranslated region (UTR) is required for preferential accumulation of GUS in M cells. This element is present and functional in three additional 5' UTRs and six 3' UTRs where it determines accumulation of two isoforms of CA and pyruvate,orthophosphate dikinase in M cells. Although the GgCA4 5' UTR is sufficient to direct GUS accumulation in M cells, transcripts encoding GUS are abundant in both M and BS. Mutating the GgCA4 5' UTR abolishes enrichment of protein in M cells without affecting transcript abundance. The work identifies a mechanism that directs cell-preferential accumulation of multiple enzymes required for C4 photosynthesis.

Methylation-sensitive expression of a DNA demethylase gene serves as an epigenetic rheostat.

Genomes must balance active suppression of transposable elements (TEs) with the need to maintain gene expression. In Arabidopsis, euchromatic TEs are targeted by RNA-directed DNA methylation (RdDM). Conversely, active DNA demethylation prevents accumulation of methylation at genes proximal to these TEs. It is unknown how a cellular balance between methylation and demethylation activities is achieved. Here we show that both RdDM and DNA demethylation are highly active at a TE proximal to the major DNA demethylase gene ROS1. Unexpectedly, and in contrast to most other genomic targets, expression of ROS1 is promoted by DNA methylation and antagonized by DNA demethylation. We demonstrate that inducing methylation in the ROS1 proximal region is sufficient to restore ROS1 expression in an RdDM mutant. Additionally, methylation-sensitive expression of ROS1 is conserved in other species, suggesting it is adaptive. We propose that the ROS1 locus functions as an epigenetic rheostat, tuning the level of demethylase activity in response to methylation alterations, thus ensuring epigenomic stability.

Arabidopsis uses two gluconeogenic gateways for organic acids to fuel seedling establishment.

Gluconeogenesis is a fundamental metabolic process that allows organisms to make sugars from non-carbohydrate stores such as lipids and protein. In eukaryotes only one gluconeogenic route has been described from organic acid intermediates and this relies on the enzyme phosphoenolpyruvate carboxykinase (PCK). Here we show that two routes exist in Arabidopsis, and that the second uses pyruvate, orthophosphate dikinase (PPDK). Gluconeogenesis is critical to fuel the transition from seed to seedling. Arabidopsis pck1 and ppdk mutants are compromised in seed-storage reserve mobilization and seedling establishment. Radiolabelling studies show that PCK predominantly allows sugars to be made from dicarboxylic acids, which are products of lipid breakdown. However, PPDK also allows sugars to be made from pyruvate, which is a major product of protein breakdown. We propose that both routes have been evolutionarily conserved in plants because, while PCK expends less energy, PPDK is twice as efficient at recovering carbon from pyruvate.

Phenotypic landscape inference reveals multiple evolutionary paths to C4 photosynthesis.

C4 photosynthesis has independently evolved from the ancestral C3 pathway in at least 60 plant lineages, but, as with other complex traits, how it evolved is unclear. Here we show that the polyphyletic appearance of C4 photosynthesis is associated with diverse and flexible evolutionary paths that group into four major trajectories. We conducted a meta-analysis of 18 lineages containing species that use C3, C4, or intermediate C3-C4 forms of photosynthesis to parameterise a 16-dimensional phenotypic landscape. We then developed and experimentally verified a novel Bayesian approach based on a hidden Markov model that predicts how the C4 phenotype evolved. The alternative evolutionary histories underlying the appearance of C4 photosynthesis were determined by ancestral lineage and initial phenotypic alterations unrelated to photosynthesis. We conclude that the order of C4 trait acquisition is flexible and driven by non-photosynthetic drivers. This flexibility will have facilitated the convergent evolution of this complex trait. DOI:http://dx.doi.org/10.7554/eLife.00961.001.

Molecular evolution of genes recruited into C4 photosynthesis.

The C4 pathway is found in 62 lineages of land plants. We assess evidence for parallel versus convergent evolution of C4 photosynthesis from three approaches: (i) studies of specific genes and cis-elements controlling their expression; (ii) phylogenetic analyses of mRNAs and inferred amino acid sequences; and (iii) analysis of C3 and C4 genomes and transcriptomes. Evidence suggests that although convergent evolution is common, parallel evolution can underlie both changes to gene expression and amino acid sequence. cis-elements that direct cell specificity in C4 leaves are present in C3 orthologues of genes recruited into C4, probably facilitating this parallel evolution. From this, and genomic data, we propose that gene duplication followed by neofunctionalisation is not necessarily important in the evolution of C4 biochemistry.

Multiple Arabidopsis genes primed for recruitment into C4 photosynthesis.

C(4) photosynthesis occurs in the most productive crops and vegetation on the planet, and has become widespread because it allows increased rates of photosynthesis compared with the ancestral C(3) pathway. Leaves of C(4) plants typically possess complicated alterations to photosynthesis, such that its reactions are compartmented between mesophyll and bundle sheath cells. Despite its complexity, the C(4) pathway has arisen independently in 62 separate lineages of land plants, and so represents one of the most striking examples of convergent evolution known. We demonstrate that elements in untranslated regions (UTRs) of multiple genes important for C(4) photosynthesis contribute to the metabolic compartmentalization characteristic of a C(4) leaf. Either the 5' or the 3' UTR is sufficient for cell specificity, indicating that functional redundancy underlies this key aspect of C(4) gene expression. Furthermore, we show that orthologous PPDK and CA genes from the C(3) plant Arabidopsis thaliana are primed for recruitment into the C(4) pathway. Elements sufficient for M-cell specificity in C(4) leaves are also present in both the 5' and 3' UTRs of these C(3) A. thaliana genes. These data indicate functional latency within the UTRs of genes from C(3) species that have been recruited into the C(4) pathway. The repeated recruitment of pre-existing cis-elements in C(3) genes may have facilitated the evolution of C(4) photosynthesis. These data also highlight the importance of alterations in trans in producing a functional C(4) leaf, and so provide insight into both the evolution and molecular basis of this important type of photosynthesis.