DNA methylation is a key chromatin modification in plant genomes that is meiotically and mitotically heritable, and at times is associated with gene expression and morphological variation. Benefiting from the increased availability of high-quality reference genome assemblies and methods to profile single-base resolution DNA methylation states, DNA methylomes for many crop species are available. These efforts are making it possible to begin answering crucial questions, including understanding the role of DNA methylation in developmental processes, its role in crop species evolution, and whether DNA methylation is dynamically altered and heritable in response to changes in the environment. These genome-wide maps provide evidence for the existence of silent epialleles in plant genomes which, once identified, can be targeted for reactivation leading to phenotypic variation.

In this review, we describe the general pattern and distribution of DNA methylation in published crop methylomes. Although all crop species studied utilize cytosine methylation, how this base modification is translated into control of gene expression, transposon regulation, and phenotypic variation is not well understood. In particular, we discuss reported roles of DNA methylation in crop developmental processes and its role in evolutionary mechanisms such as whole-genome duplications. We also investigate the possible role of DNA methylation in the response by plants to changes in the environment, and discuss the advantages of modifying DNA methylation states for the generation of novel variation useful to breeding programs.

Although genetic and genomic studies of DNA methylation in A. thaliana have made scientific advances in the field, how these results transfer to crop species is unclear. Multiple studies in a range of crop species have reported the role of DNA methylation in controlling gene expression and resulting morphological variation (), highlighting the importance of studying DNA methylation in these species. To date, methylomes of eight crop species have undergone genome-wide sequencing using WGBS ( Table 1 ), including Brassica napus (oilseed rape) (), Brassica oleracea (), Glycine max (soybean) (), Oryza sativa (rice) (), Populus trichocarpa (black cottonwood) (), Solanum lycopersicum (tomato) (), Sorghum bicolor (sorghum) (), and Zea mays (maize) (). If we extend the scope to include non-crop species, the total number increases to 14, including Amborella trichopoda (Amborella) (), Arabidopsis lyrata (), A. thaliana (), Brachypodium distachyon (Brachypodium) (), and Capsella rubella ().

Gene body methylation shows distinct patterns associated with different gene origins and duplication modes and has a heterogeneous relationship with gene expression in Oryza sativa (rice).

List of DNA Methylomes and Epigenomic Studies Published throughout the Plant Kingdom and Their Associated References (Arabidopsis thaliana Is Not Listed).

Table 1 List of DNA Methylomes and Epigenomic Studies Published throughout the Plant Kingdom and Their Associated References (Arabidopsis thaliana Is Not Listed).

Gene body methylation shows distinct patterns associated with different gene origins and duplication modes and has a heterogeneous relationship with gene expression in Oryza sativa (rice).

WGBS has led to a number of discoveries and novel fields of research in both plant and animal species. For example, human DNA methylation patterns differ between specific cell types and show methylation in two distinct contexts (). Methylation in a CG context occurs ubiquitously, whereas methylation in a CH (H = A, T, C) context occurs in brain and embryonic stem cells (). In contrast to humans, DNA methylation in plants is separated into three distinct contexts: CG, CHG, and CHH. Each of these require different mechanisms for their establishment/maintenance and have different effects on their target DNA sequences (). In A. thaliana, CG methylation is maintained by the DNA methyltransferase MET1 () whereas CHG and CHH is controlled by methylation of histone H3 lysine 9 (H3K9) and maintained by the methyltransferases CMT3 and CMT2, respectively (). Regions of the genome methylated in all three contexts often lead to silencing in the targeted region and in some cases, neighboring regions (). The RNA-directed DNA methylation pathway (RdDM) requires the activities of the DNA methyltransferase DRM1/DRM2 () and is one of the primary mechanisms for non-CG methylation (). In A. thaliana, this pathway is guided by 24 nucleotide short interfering RNAs (siRNAs) derived from transcripts generated by the plant-specific RNA polymerase Pol IV (). DNA methylation-induced silencing is mostly found in transposable elements (TEs), repeat sequences, and some genes (). In contrast, protein-coding genes methylated in only the CG context are referred to as “gene-body methylated” (). Although its functions remain unclear, many studies of plant genomes reveal that this type of methylation is enriched in exonic regions and depleted of siRNAs (). Furthermore, these genes have moderately high expression levels and tend to be more stably expressed, being less responsive to environmental and developmental cues ().

Methodologies to explore the patterns of DNA methylation in genomes have rapidly advanced during the last 10 years. Initially, methylation-sensitive restriction digestion methods were used to profile genome-wide distributions of DNA methylation (). Taking advantage of the sensitivity of certain restriction enzymes’ ability to digest methylated DNA, methods were developed to visualize differential methylation after methylation-sensitive restriction digestions with electrophoresis, microarray hybridizations, and other detection methods (). However, this general approach is limited by the sites that each enzyme digests and the distribution of each enzyme’s target motif, and cannot determine DNA methylation states within each fragment, thus giving a relatively low-resolution method for the detection of DNA methylation within the genome. Following this method was the development of immunoprecipitation-based assays (). 5-Methylcytosine (5mC) specific antibodies are used to immunoprecipitate methylated cytosines () and then are coupled with gene-chip or high-throughput sequencing (MeDIP-chip or MeDIP-seq), which improves the resolution at which DNA methylation can be detected to dozens of base pairs (). However, this approach is restricted by each antibody’s specificity and is heavily dependent on the distribution of 5mC throughout the genome (). In 2008, the emergence of sodium bisulfite conversion combined with high-throughput sequencing (whole-genome bisulfite sequencing [WGBS]) led the field into the single-base resolution era, and significantly advanced our understanding of how genomes utilize DNA methylation (). The principle of this technique is to use a sodium bisulfite reaction to convert unmethylated cytosines to uracils, which are then converted to thymines during PCR amplification (). High-resolution sequencing of the converted DNA is then compared with a reference genome, allowing determination of the methylation status of every cytosine in the genome at a single-base resolution, as methylated cytosines are protected from conversion by this reaction and read as cytosines after sequencing ().

Genome-wide studies have undergone an unprecedented advancement following the increased availability of high-quality reference genome assemblies. The first plant reference genome was generated for Arabidopsis thaliana in 2000 (); since then, more than 40 high-quality plant reference genomes have been published (). Utilizing these publicly available reference genomes, the first genome-wide maps of DNA methylation were produced for a number of plant species revealing that all studied plants use DNA methylation (), but not necessarily in exactly the same way (). DNA methylation is one of the most extensively studied chromatin modifications in plants (). It can be meiotically and mitotically heritable () and is one mechanism that provides a link between morphological variation and the DNA sequence. This base modification is involved in a number of plant processes, as changes in DNA methylation states are often associated with alteration to gene expression and resulting phenotypes ().

Using chromatin immunoprecipitation sequencing (ChIP-seq) of RIPENING INHIBITOR (RIN), a key ripening transcription factor, an association was identified with the promoters of numerous genes important for fruit ripening (). The function of RIN in fruit ripening is dependent on CNR (). The cnr mutant used in these studies is an epiallele with a hypermethylated promoter that reduces CNR gene expression (). A genome-wide analysis of methylation patterns throughout fruit development demonstrated many RIN targeted genes fail to undergo demethylation in the cnr mutant, indicating that CNR is required for the changes in DNA methylation of RIN targeted loci and explaining the lack of transcriptional activity for these genes ( Figure 2 ) (). This comprehensive study demonstrates the complexity, and necessity, of the dynamic methylation changes important to fruit ripening in tomato (). Collectively, studies from endosperm tissue and the study from tomato provides evidence for the role of methylation in certain developmental processes () and also demonstrates the need for technologies and methodologies that enrich for specific cell types to increase the resolution and detection of methylation changes genome wide.

Evidence from a comprehensive epigenomic study in tomato provides another example of the potential function of DNA methylation to influence a plant developmental process. Many crop species have fleshy fruits, which is a differentiated floral tissue that evolved to aid seed dispersal in some flowering plants. Plants with this trait undergo a distinctive development stage known as fruit ripening following seed maturation, in which the fruit undergoes numerous physical changes to attract seed dispersal vectors (). The plant hormone ethylene is involved in controlling the onset of fruit ripening, but it is only effective after fruit seeds first become viable, implying that other factors exist (). A genome-wide analysis of DNA methylation throughout a developmental time course in a carefully dissected tomato fruit pericarp tissue demonstrated that multiple ripening-related genes including the transcription factor CNR (COLORLESS NON-RIPENING), the pectinase polygalacturonase PG2A (POLYGALACTURONASE 2A), and the rate-limiting enzyme in fruit carotenoid synthesis PSY1 (PHYTOENE SYNTHASE) possess hypermethylated 5′ regions prior to fruit ripening (). However, these regions become demethylated and undergo active transcription during ripening stages ( Figure 2 ). Furthermore, early introduction of the methyltransferase inhibitor 5-azacytidine caused premature demethylation in many of these genes and induced early fruit ripening ().

In immature fruit, numerous genes associated with ripening have methylated promoters, which inhibit RIN targeting and subsequent transcriptional activation. In an uncharacterized manner, the promoter regions become demethylated during ripening stages and corresponding genes experience binding by RIN and active transcription, triggering fruit ripening. The cnr mutant inhibits the demethylation process and prevents fruit ripening.

In the rice endosperm, genome-wide demethylation was observed in non-CG contexts associated with short euchromatic TEs, but demethylation within CG contexts was localized to only 7% of loci (), which is similar to previous findings in A. thaliana (). The observed loss in non-CG methylation in rice is proposed to reactivate genes normally silenced in the sporophyte. Further investigation revealed that 165 demethylated genes were preferentially expressed in the endosperm, including genes responsible for synthesizing starch proteins, which are confined to the endosperm, providing support for this tissue-specific gene activation process (). In A. thaliana, the demethylation in endosperm is mediated by the DNA glycosylase DME (DEMETER) (), but an ortholog is not present in rice (). Instead, in rice it appears that ROS1a (REPRESSOR OF TRANSCRIPTIONAL GENE SILENCING1a), a homolog of DME, likely carries out this function ().

The lack of variation found in the soybean and sorghum studies can most likely be explained by the complexity of the tissues studied, although it should be noted that changes in DNA methylation may not be required for many plant developmental processes. These studies searched for variation between tissue types, which possess a large diversity of cell types. For instance, the only significant intra-individual variation found between the two studies was for the endosperm, a tissue composed of just a few cell types. Future studies of genome-wide patterns of DNA methylation using a single cell or single cell type will likely have greater success in elucidating the role of DNA methylation in plant development ( Figure 1 ). For example, studies in rice, tomato, and maize have already demonstrated increased ability to detect genome-wide changes to DNA methylation by using methods to reduce the number of cell types being profiled ().

High-throughput sequencing provides unprecedented resolution of DNA methylomes and transcriptomes. However, a major challenge for studying single-base resolution changes in DNA methylation controlled by the environment or by development is cellular complexity associated with most experiments. Whereas multiple transcripts are produced from a single gene, there is only a single genomic region associated with each gene for each chromosome. The amplification of abundant transcripts from a single locus increases the ability to detect differentially expressed genes in complex cell populations compared with detection of differentially methylated regions. Methods to overcome these challenges include enriching specific cell types or sequencing epigenomes of single cells. Furthermore, increasing the sequencing depth increases statistical power to detect differentially methylated regions.

A comprehensive understanding of the mechanisms underlying the diverse growth and development of plants to a harvestable product is essential in generating better crop cultivation and breeding methodologies. Previous studies indicate that certain developmental processes are influenced by changes in DNA methylation. Two recent studies looked for evidence for variation in DNA methylation between organ and tissue types in soybean and sorghum, respectively ().produced single-base resolution methylomes for four organ types at 13× coverage in soybean. Although they found little difference between methylation status of organs, they did find support for rare hypomethylation events associated with higher gene expression levels of nearby genes. However, whether these were spontaneous in nature or were developmentally controlled differentially methylated regions (DMRs) is unclear. Sorghum tissue methylation profiles produced at lower resolution using methylation-sensitive amplified polymorphism revealed similar findings, with insignificant methylation changes across seven tissues except for the endosperm (). The endosperm possessed genome-wide hypomethylation, which concurs with studies on A. thaliana, rice, and maize endosperm ().

The Role of DNA Methylation in the Evolution of Plant Genomes

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Gaut B.S. Body-methylated genes in Arabidopsis thaliana are functionally important and evolve slowly. Results from sequencing DNA methylomes from insects, algae, plants, and animals confirmed that gene body methylation is not exclusive to plants (). To explore the conservation of gene body methylation, a phylogenetic analysis of enzymes involved in DNA methylation found that UHRF1 (ubiquitin-like with PHD and RING finger domains 1) and its homologs exist in all species with CG methylation, but is absent from species lacking CG methylation (). It should be noted that although numerous genes in animals and plants contain CG methylation in the gene bodies, the gene body methylation found in plants that is associated with moderately expressed genes that are slowly evolving is not found in animals. Therefore, it is unlikely that the function and evolutionary consequence of gene body methylation between plants and animals is the same. Finally, the function of gene body methylation in plant genomes is also unclear, although it has been speculated that it may simply be a by-product of transcription or other nuclear processes ().