The Role of Plant DNA Methylation in Development, Stress Response, and Crop Breeding

Abstract

DNA methylation, an evolutionarily conserved epigenetic mechanism, is crucial for controlling gene activity and ensuring genomic integrity. Altered methylation patterns can profoundly affect plant development, often resulting in atypical phenotypes. The regulation of these methylation states relies on the coordinated actions of de novo methylation, maintenance, and active demethylation, orchestrated by specialized enzymes within distinct pathways. This review delves into the diverse roles of DNA methylation in plants, offering an in-depth analysis of the enzymes and regulatory factors involved. We explore how these elements function within the broader epigenetic framework, focusing on their contributions to silencing transposable elements, modulating gene expression, and shaping chromatin architecture. The review also examines the significance of DNA methylation in plant development, particularly its role in adapting to biotic and abiotic stresses. Lastly, we highlight its potential for driving innovations in crop breeding, emphasizing its applicability in advancing sustainable agriculture.

1. Introduction

Epigenetic modifications represent heritable chemical changes that influence phenotypic traits without altering the underlying DNA nucleotide sequence [1,2]. In plants, the epigenome’s dynamic and plastic nature is crucial for regulating development, environmental adaptation, and evolutionary processes [3]. Key epigenetic marks encompass DNA methylation and diverse post-translational modifications of histones [4,5,6]. Additionally, non-coding RNAs and chromatin remodeling factors play integral roles in modulating gene expression and genome accessibility, thereby facilitating phenotypic plasticity [7]. Collectively, these epigenetic mechanisms enable plants to rapidly adapt to fluctuating environmental conditions, underscoring their evolutionary significance.

DNA methylation is a fundamental and extensively studied epigenetic mechanism in eukaryotes. Often referred to as 5-methylcytosine (5mC), it plays pivotal roles in transposon silencing, transcriptional regulation, and the maintenance of normal growth and development in numerous organisms [8,9,10]. Notably, plant genomes consistently exhibit extensive DNA methylation, with 5mC constituting up to 50% of total cytosine residues in some species [11]. This methylation occurs across all sequence contexts (CG, CHG, and CHH, where H represents A, C, or T). Euchromatin refers to loosely packed chromatin associated with transcriptionally active genes. DNA methylation within euchromatin predominantly occurs at CpG islands, modulating gene expression by either promoting activation or repressing transcription. Methylation levels are typically low to moderate, serving a dynamic role in the precise regulation of gene activity. This modification is frequently associated with active histone marks, such as H3K4me3, which facilitate transcriptional processes. In contrast, heterochromatin is tightly packed and harbors transcriptionally silent regions, including repetitive DNA and centromeres. DNA methylation in heterochromatin is generally elevated, contributing to gene silencing and the maintenance of genomic stability. This form of methylation is often linked to repressive histone marks, such as H3K9me3, and plays a key role in silencing transposable elements (TEs) and other repetitive sequences. Furthermore, many genic regions in plant genomes are methylated at CG sites, although this methylation is not directly linked to transcriptional gene silencing [12]. Despite these insights, the broader functional implications of genomic methylation remain incompletely understood and warrant further investigation.

DNA methylation, a conserved epigenetic modification shared by plants and animals, plays a crucial role in regulating development by maintaining specific genomic patterns. This modification is facilitated by DNA methyltransferases, which are highly conserved across both kingdoms and utilize S-adenosyl-L-methionine as a methyl donor. Conversely, active demethylation occurs through the base excision repair pathway, reflecting the dynamic nature of methylation [13,14,15,16]. In Arabidopsis thaliana, five DNA methyltransferases—METHYLTRANSFERASE 1 (MET1), CHROMOMETHYLASE 2 (CMT2), CMT3, and DOMAINS REARRANGED METHYLTRANSFERASE 1 (DRM1) and DRM2—are essential for establishing and maintaining methylation patterns [17]. MET1 specifically regulates CG methylation by interacting with methyl-CG-binding proteins VIM1, VIM2, and VIM3, ensuring semi-conservative methylation during DNA replication [18]. CHG methylation is primarily maintained by CMT3 through a positive feedback loop involving histone H3 lysine 9 mono- and dimethylation (H3K9me1/2) [19]. Similarly, CMT2 facilitates CHH methylation, particularly in heterochromatic regions, using mechanisms akin to those of CMT3 [19]. DRM1 and DRM2, with DRM2 as the key enzyme, are responsible for CHH methylation via the RNA-directed DNA methylation (RdDM) pathway, which relies on non-coding RNAs for de novo methylation in plants [20]. Beyond its role in RdDM, DRM2 is the principal enzyme for de novo methylation across all sequence contexts, underscoring its central role in epigenetic regulation (Figure 1).

This review examines recent advances and current perspectives on the regulation and function of DNA methylation in plants. The mechanisms that establish specific DNA methylation patterns are most comprehensively characterized in the model plant Arabidopsis thaliana, where mutations in DNA methylation and demethylation pathways, as well as alterations in regulatory factor composition, typically do not lead to plant lethality. In contrast, for plants with more complex genomes, DNA methylation is increasingly recognized as a critical regulator of development and adaptation to environmental stress. Emerging research has uncovered pivotal mechanisms governing plant DNA methylation. Notable examples include the initiation of de novo DNA methylation mediated by non-coding RNAs (ncRNAs), the activity of the novel protein complex IDM, which targets active DNA demethylation and paradoxically enhances DNA methylation, and the regulatory role of methylation-sensing genetic elements that dynamically balance methylation and demethylation processes. Additionally, this review highlights the dynamic roles of DNA methylation in regulating various biological processes, including transposon silencing, gene expression, chromosomal interactions, plant development, and responses to both biotic and abiotic environmental stimuli. Finally, the potential applications of DNA methylation dynamics in crop improvement and breeding are discussed, emphasizing its transformative potential in agricultural innovation.

2. DNA Methylation Kinetics

2.1. Establishment of DNA Methylation

In plants, de novo DNA methylation is mediated by the RdDM pathway, which involves small interfering RNAs (siRNAs), scaffold RNAs, and associated proteins. In Arabidopsis thaliana, the canonical RdDM pathway begins with RNA polymerase IV (Pol IV), which transcribes a precursor RNA that is then converted into double-stranded RNA (dsRNA) by RNA-dependent RNA polymerase 2 (RDR2) [20,21,22]. Dicer-like protein 3 (DCL3) processes this dsRNA into 24-nucleotide siRNAs, which are incorporated into ARGONAUTE (AGO) proteins, primarily AGO4 [23,24]. These siRNAs guide the recognition of complementary scaffold RNAs transcribed by RNA polymerase V (Pol V). AGO4 interacts with the DNA methyltransferase DRM2 to catalyze de novo DNA methylation in a sequence-independent manner [25,26,27]. RdDM is also involved in the regulation of parasitic sequences, such as TEs and viruses, where foreign RNAs are converted into dsRNA and processed into siRNAs by RNA-dependent RNA polymerases like RDR6. These siRNAs guide DNA methylation through AGO proteins, although the mechanisms of non-canonical RdDM remain poorly understood [28,29,30].

Scaffold RNAs, produced by Pol V, are retained on chromatin through interactions with chromatin-remodeling factors such as ribosomal RNA processing 6-like 1 (RRP6L1), a homolog of yeast RRP6. These RNAs, along with siRNAs, are stabilized by the INVOLVED IN DE NOVO 2 (IDN2-IDN2) paralogue complex and the SWI/SNF chromatin remodeling complex, facilitating Pol V-mediated transcriptional silencing [31,32,33,34]. Furthermore, histone modifications also play a critical role in RdDM. Six histone H1 (SHH1), a histone-binding protein, recruits Pol IV to chromatin at specific loci, while zinc finger, mouse double-minute/switching complex B, Plus-3 protein (ZMP) regulates Pol IV binding to pericentromeric regions [35,36,37]. The CLASSY family of chromatin remodelers is involved in Pol IV-dependent siRNA production, and the DDR complex, which includes D(1A) dopamine receptor (DRD1) and DMS3, is essential for Pol V-mediated RNA transcription [38,39,40,41,42]. Pol V generates non-coding RNAs (ncRNAs), which lack polyadenylation and are typically ~200 nucleotides long. These ncRNAs can initiate transcription without a promoter and are capped with 7-methylguanosine [43,44,45,46,47,48]. The siRNAs produced in the canonical pathway can also be generated through non-canonical routes involving RNA polymerase II (Pol II), which produces 24-nt siRNAs and recruits Pol IV and Pol V for RdDM [49,50,51,52]. In some regions, RdDM relies on RDR6 and Pol II, with DCL2 and DCL4 generating shorter siRNAs (21–22 nt), suggesting an alternative, DCL-independent RdDM mechanisms [53,54].

Overall, while much is understood about the canonical RdDM pathway (Figure 2), alternative pathways and interactions with RNA processing factors continue to present challenges in fully delineating the molecular mechanisms of RdDM [55,56,57].

2.2. Maintenance of DNA Methylation

In Arabidopsis thaliana, maintaining DNA methylation patterns relies on the coordinated actions of five DNA methyltransferases. CHG and CHH methylation are preserved through self-reinforcing feedback systems that involve CMT3, CMT2, DRM1, DRM2, histone methyltransferases, and nucleosomes modified with repressive marks such as H3K9me1 and H3K9me2 [58,59]. CG methylation, however, is predominantly maintained by MET1, which is believed to methylate hemimethylated CG sites during DNA replication, ensuring methylation continuity on newly synthesized DNA strands [59,60]. Research on DNMT1, the mammalian homolog of MET1, shows a preference for hemimethylated DNA substrates both in vitro and in vivo, aligning with this semi-conservative methylation model [61,62,63].

During replication, DNMT1 and the E3 ubiquitin ligase UHRF1 are localized to replication foci, a process critical for preserving CG methylation in mammals [64,65]. In plants, UHRF1 homologs are encoded by the VIM gene family, whose role in CG methylation parallels their mammalian counterparts. Notably, CG methylation is abolished in Arabidopsis mutants lacking MET1 or in triple mutants of VIM1, VIM2, and VIM3 [66]. Both DNMT1 and UHRF1 are multi-domain proteins, with catalytic domains located at their C-terminus and regulatory domains at the N-terminus [67]. Interestingly, the N-terminal domains of MET1 and VIM proteins diverge structurally from those of DNMT1 and UHRF1, indicating that plants and mammals utilize distinct regulatory strategies (Figure 3).

2.3. Demethylation of Active DNA

The failure to maintain DNA methylation following replication, due to the absence of either DNA methyltransferase activity or a methyl donor, results in passive DNA demethylation—a process distinct from enzymatic removal of methylation marks known as active DNA demethylation [68,69,70]. In plants, active DNA demethylation is initiated by a family of bifunctional 5-mC DNA glycosylase-purine/depyrimidine lyases, which excise 5-mC through the base excision repair (BER) pathway [71,72]. Similarly, active demethylation in mammals involves DNA glycosylases and the BER mechanism. However, a key distinction lies in substrate recognition: plant DNA glycosylases directly identify and excise 5-mC bases, whereas in mammals, 5-mC undergoes oxidation prior to glycosylase-mediated excision [73] (Figure 4).

Arabidopsis thaliana contains a group of four bifunctional 5-mC DNA glycosylases: REPRESSOR OF SILENCING 1 (ROS1), DEMETER (DME), and the DEMETER-LIKE PROTEINS DML2 and DML3 [71,73]. These glycosylases actively remove 5-mC from cytosines in all sequence contexts [72,74,75,76]. While ROS1, DML2, and DML3 are widely expressed in vegetative tissues, DME shows a more restricted expression profile, being primarily active in the companion cells of male and female gametophytes. Specifically, DME is expressed in the central cells of female gametophytes and in the trophic cells of male gametophytes [75,77].

During DNA demethylation, bifunctional enzymes initially function as DNA glycosylases, hydrolyzing the glycosyl bond between the base and deoxyribose. Subsequently, they act as apurinic/apyrimidinic (AP) lyases, cleaving the DNA backbone to generate abasic (base-free) sites. Following the excision of the 5-methylcytosine (5-mC) base, either a β-elimination or β, δ-elimination reaction occurs. These reactions create a gap in the DNA strand, terminating with either a 3′-phosphate-α, β-unsaturated aldehyde or a 3′-phosphate, respectively. In the downstream processing of these elimination reactions, AP endonuclease 1 (APE1) and the polynucleotide 3′-phosphatase ZDP play crucial roles. APE1 operates following β-elimination, while ZDP acts after β, δ-elimination, both facilitating the generation of a 3′-hydroxyl (3′-OH) group. This modification prepares the DNA strand for subsequent repair by DNA polymerases and ligases, which restore the integrity of the DNA [78,79,80].

3. Molecular Functions of Plant DNA Methylation

3.1. Regulation of Gene Expression

In plants, DNA methylation associated with genes typically occurs in promoter regions or transcribed areas of the genome (Figure 5A,B). Promoter methylation is generally linked to transcriptional suppression, though it can occasionally promote gene activation. This dual functionality has been documented in the regulation of the ROS1 gene in Arabidopsis and in numerous genes that inhibit tomato fruit ripening [18,81,82,83]. Transcriptional repression by promoter methylation involves both direct and indirect pathways. Directly, it may block the binding of transcriptional activators or enhance the interaction with repressors. Indirectly, it contributes to transcriptional silencing by promoting repressive histone modifications, such as H3K9me2, and reducing activating modifications like histone acetylation [84,85].

The exact mechanism by which promoter methylation activates transcription remains unclear. One theory posits that methylation might enhance the binding of specific transcriptional activators or impede the attachment of repressors. Promoter methylation frequently originates from the expansion of methylation from adjacent transposable elements (TEs) or repetitive sequences. To protect neighboring genes from being silenced, these TEs and repeats are often subjected to active DNA demethylation [86]. Notably, for genes requiring promoter methylation for expression, active demethylation may unexpectedly result in transcriptional repression. This dynamic interplay between DNA methylation and gene expression underscores the complexity of transcriptional regulation [82,87].

Promoter methylation relies on various effectors, including methyl-DNA-binding proteins, histone modifiers, chromatin remodeling factors, and molecular chaperones, to establish a condensed chromatin structure that limits transcription factor access. In Arabidopsis, chaperones play a notable role in modulating chromatin states enriched in mCG sequences, particularly in promoter regions [88,89,90]. Proteins such as MBD7, MBD5, and MBD6, which recognize mCG sites, contain a C-terminal StkyC domain that interacts with ACD family chaperones [88,90]. ACD chaperones, including IDM2 and IDM3, facilitate the recruitment of histone acetyltransferase IDM1 and accessory proteins HDP1 and HDP2, forming IDM complexes essential for demethylating and activating transcription at the highly methylated viral 35S promoter [90,91].

At another 35S promoter locus, ACD proteins like RDS1, RDS2, and IDM3 recruit additional chaperones, such as DnaJ domain proteins SDJ4 and HSP70, to assemble molecular chaperone complexes. These complexes can modulate the transcriptional state of the locus depending on the genetic context, either silencing or activating it [88]. Despite these advances, the precise functions of chaperone proteins in methylated loci require further investigation.

Interestingly, MBD proteins possess intrinsically disordered regions, which may facilitate phase separation to form molecular aggregates. ACD proteins, known as “maintainers”, bind target proteins to prevent irreversible aggregation [92]. DnaJ domain proteins, often working with HSP70, contribute to ATP-dependent chaperone activity that supports substrate protein refolding or depolymerization [92]. This regulatory interplay between chaperone proteins and MBD proteins may influence the aggregation state of MBDs, thereby controlling chromatin accessibility at mCG-rich loci.

3.2. TEs Silence

By relocating transposons (TE) or inserting new retrotransposon copies, TE poses a significant threat to genomic stability. In Arabidopsis, the heterochromatin regions around the centromeres, as well as certain non-heterochromatin regions containing TE or repeat sequences, exhibit extensive methylation in all cytosine environments [93] (Figure 5C). Rna-directed DNA methylation (RdDM) plays a key role in maintaining asymmetric CHH methylation at the edges of short and long TE. In the internal region of long heterochromatin transposons, asymmetric methylation is regulated by chromatin remodeling factor DDM1 and catalyzed by CMT2 [19,94]. In the maize genome, active genes, and inactive TE are staggered, often separated by RDDM-dependent “islands” of CHH methylation. These methylated islands are short genomic regions with high levels of CHH methylation. Loss of these islands often results in transcriptional activation, accompanied by decreased methylation levels at CG and CHG sites in adjacent TE. This suggests that in maize, RdDM is critical for silencing transposons in costained regions containing adjacent active genes [95]. In sugar beets, CHH methylation appears to be more specifically involved in the silencing of DNA transposons. DNA transposons show higher levels of CHH methylation compared to retrotransposons and genes [96]. The CG and CWG (a subset of CHG) motifs are symmetric, while the CHH motifs are asymmetric. In partially symmetric CCG motifs, the methylation of the first cytosine is dependent on CG methylation (citation), so CCG methylation levels are generally lower than CWG methylation [97]. In addition, in monocotyledonous plants such as maize and rice, environment-specific CHH methylation is more evenly distributed on chromosomes, further indicating the diversity of its regulatory mechanisms [97].

3.3. Chromosome Interaction and Biogenesis of circRNAs

DNA methylation shapes the epigenetic state of chromatin and is pivotal in regulating chromosomal interactions. In the nucleus of Arabidopsis thaliana, all five chromosomes interact through a specialized structure called the KNOT [98]. This structure consists of interactive heterochromatin islands (IHIs), which are repressive chromatin regions embedded within euchromatin arms and enriched with transposable elements (TEs) [98,99]. Interestingly, the integrity of IHI interactions remains intact in met1 and ddm1 mutants, despite extensive DNA hypomethylation across all cytosine contexts. Similarly, these interactions are unaffected in the suvh4-suvh5-suvh6 triple mutant, which lacks functional H3K9 methylation [99]. These observations indicate that both DNA methylation and H3K9me2 are indispensable for preserving chromosomal interactions within IHIs (Figure 5D).

Interestingly, mutants deficient in RdDM display increased frequencies of chromosomal interactions in specific RdDM-targeted regions. This indicates that, in wild-type plants, RdDM acts to suppress chromosome interactions in certain genomic regions [100]. Moreover, enhanced chromosomal interactions are observed between POL V-dependent DNA methylation sites and distal genes that are repressed by RdDM. These findings suggest that chromosomal interactions may also play a regulatory role in gene expression [100]. Furthermore, some studies have suggested that there may be synergies between DNA methylation and long non-coding Rnas (lncrnas) or micrornas (mirnas) that co-regulate circRNA synthesis and degradation (Figure 5D) [100].

4. The Role of DNA Methylation in Plant Development

4.1. Genomic Imprinting and Seed Development

Arabidopsis utilizes a specialized double fertilization mechanism, which depends on the multicellular structure of both male and female gametophytes. In this process, the two sperm cells carried within a single pollen grain fertilize separate cells of the female gametophyte: one sperm cell fuses with the egg cell to form the embryo, while the other combines with the central cell to develop the endosperm. In both rice and Arabidopsis thaliana, the endosperm exhibits global DNA hypomethylation compared to the embryo [101,102,103]. In A. thaliana, this hypomethylation is attributed in part to active demethylation mediated by DEMETER (DME) in pre-fertilization central cells, which are the partner cells of female gametes [101,103]. Interestingly, although MET1 transcription is repressed during female gametogenesis, this repression does not appear to drive extensive demethylation. Consistent with this observation, genome-wide CG hypomethylation, which would be expected under reduced MET1 activity, is not detected in wild-type endosperm. Moreover, DNA methylation is almost entirely restored in the endosperm of dme mutants, underscoring the critical role of DME-dependent pathways in establishing endosperm-specific epigenetic patterns [102,104].

DME-mediated demethylation occurs in androtrophic cells and is accompanied by the downregulation of DDM1 [105]. This process triggers the production of small interfering RNAs (siRNAs) derived from demethylated TEs, which are subsequently transported to sperm cells, where they enhance RNA-directed DNA methylation (RdDM). These transposon-derived siRNAs in sperm cells may also contribute to transposon silencing in egg cells after fertilization [105,106,107]. The levels of CHH methylation dynamically fluctuate during seed development and germination. Specifically, CHH methylation increases during seed development but decreases during germination, primarily due to passive demethylation, suggesting its potential role in regulating seed dormancy [108]. Notably, while male reproductive lineages exhibit lower overall CHH methylation than somatic cells, certain hypermethylated CHH sites are essential for meiosis [109]. In the endosperm, methylation levels of the maternal genome are lower than those of the paternal genome, particularly at CG sites, which are closely associated with gene imprinting. Maternal expression genes (MEGs) typically exhibit hypomethylation of maternal alleles and hypermethylation of paternal alleles. For instance, certain MEGs, such as MEDEA, are silenced not through DNA methylation but via histone modification by H3K27me3 [109,110,111,112]. Disruptions in the maternal function of DME or the paternal function of MET1 interfere with the imprinting of MEGs, underscoring the crucial role of DNA methylation in allele-specific regulation [109,113,114,115]. Conversely, the maternal alleles of paternally expressed genes (PEGs) are typically marked by H3K27me3, while the paternal alleles are expressed via H3K36me3. This coordinated regulation by histone modifications and DNA methylation establishes the distinct expression patterns of MEGs and PEGs in the endosperm [116,117].

4.2. Role of DNA Methylation in Plant Meristem and Leaf Epidermal Development

Plant meristem stem cells are fundamental to tissue and organ development, with RdDM factors exhibiting higher transcription levels in meristematic tissues compared to differentiated tissues. Notably, the highest DNA methylation levels are observed in the small columnar cells of the root meristem, potentially due to reduced exposure of pericentromeric chromatin to RdDM factors [118]. While RdDM mutants in Arabidopsis do not exhibit pronounced meristem defects, corresponding mutants in rice and maize display severe developmental abnormalities, underscoring the critical role of RdDM in meristem development [118,119]. In rice, SDG711-mediated H3K27me3 deposition represses the expression of leaf development genes following shoot apical meristem development. This repression coincides with DRM2-catalyzed non-CG DNA methylation, suggesting a coordinated regulatory mechanism. Furthermore, physical interactions between SDG711 and DRM2 imply a synergistic effect in gene silencing [120]. In maize, differential regulation of maintenance DNA methyltransferases establishes distinct DNA methylation patterns across the division, transition, extension, and maturation zones of leaves. These methylation changes predominantly occur near genes involved in growth and cell cycle regulation, where promoter methylation is inversely correlated with gene expression [121]. DNA methylation also plays a pivotal role in leaf epidermal patterning in Arabidopsis. Dysfunction of ROS1 leads to hypermethylation of the EPF2 promoter, suppressing the expression of stomatal inhibitory factors and resulting in excessive stomatal production [122]. Similarly, IBM1 dysfunction enhances H3K9me2 and CHG methylation, inhibiting EPF2 receptor gene expression and disrupting stomatal patterning. Interestingly, ROS1 mutation-induced abnormalities can be alleviated by RdDM factor mutations, whereas IBM1 mutation-induced defects are mitigated by SUVH4 or CMT3 mutations. These findings suggest that DNA methylation governs leaf epidermal development through distinct regulatory pathways [123].

4.3. Role of DNA Methylation in Flower Development

DNA methylation has been shown to play a pivotal role in various aspects of flower development, including anthocyanidin deposition and floral patterning. For instance, DNA methylation influences anthocyanin accumulation in the orchid Oncidium. In Oncidium Gower Ramsey (GR), the lips of the flowers appear bright yellow due to the accumulation of carotenoid pigments. The genetic pathway responsible for this pigmentation is well characterized, and its variation accounts for the white coloration observed in the Oncidium variety known as ‘White Jade’ (WJ) [124]. The distinct color patterns observed between these orchid varieties arise from differential methylation levels of carotenoid-related genes. Specifically, in GR, the OgCCD1 gene is methylated and silenced. In contrast, in WJ tissues, the absence of methylation in the OgCCD1 promoter results in its active expression, leading to the degradation of carotenoids and the development of white flowers [125]. Another notable feature of the pigmentation pattern in GR flowers is the presence of red stripes in the perianth, which are absent in the Oncidium Honey Dollp (HD) cultivar. In this case, the contrasting flower colors are determined by the methylation status of genes involved in anthocyanin biosynthesis. In the HD variety, methylation of the OgCHS gene promoter prevents anthocyanin accumulation, whereas, in GR, unmethylated genes are expressed, facilitating anthocyanin production [126]. Additionally, DNA methylation plays a significant role in floral color formation in many other ornamental plants. For example, in Malus halliana, the color of the petals changes from red to pale pink during development, which is linked to the downregulation of several genes. Notably, the promoter of the MhMYB10 gene, which is involved in anthocyanin biosynthesis, is highly methylated, leading to reduced expression of MhMYB10 and a consequent decrease in anthocyanin accumulation [127].

In addition, DNA methylation plays a crucial role in regulating floral production. In Chinese and Japanese Prunus mume, multiple genes involved in eight floral biosynthetic pathways exhibit different methylation levels during flower development [128]. These genes code for enzymes that are key regulators of floral production, including coniferol acetyltransferase (PmCFAT1a/1c) and benzyl acetyltransferase (PmBEAT36/3). CFAT protein belongs to the acyltransferase family and is an important component of eugenol synthesis, responsible for catalyzing the conversion of coniferol-to-coniferol acetate [116]. BEAT proteins produce benzyl acetate by transferring the acetyl group from acetyl-CoA to the carbonyl group of benzyl alcohol [117]. Genome-wide sulfite sequencing showed that most of the differentially methylated genes were involved in multiple links of the benzoylalanine biosynthesis pathway, which produces more than 90% of floral volatiles [128].

4.4. Role of DNA Methylation in Fruit Ripening

During the development of tomato fruit, about 1% of the DNA methylation groups in the peel are changed. Active DNA demethylation occurs on many genes associated with fruit ripening whose promoter regions contain binding sites for ripening inhibitors (Rins). Rins are a major mature transcription factor [129]. In most known mature genes, RIN binds to the target promoter, and the expression of these genes is negatively correlated with the level of DNA methylation in the promoter region. Treatment with chemical inhibitors of DNA methylation can induce hypomethylation of the promoter region and activate the expression of the encoding colorless immature gene (CNR). CNR is a key rin target gene during fruit ripening, and this treatment can also lead to the early ripening of tomato fruits [129]. In ripe fruit of tomato (Solanum lycopersicum), expression of DNA demethylase DME-LIKE 2 (DML2) is significantly increased, mediating progressive DNA demethylation that occurs during fruit ripening [130]. Tomato DML2 acts not only on ripening inducer genes, but also on ripening suppressor genes, indicating that both activation of ripening inducer genes and inhibition of ripening suppressor genes depend on active DNA demethylation [131]. Changes in DNA methylation may be related to growth and ripening processes in other fruits. In apple fruit, anthocyanin accumulation was negatively correlated with DNA methylation levels in the apple MYB10 gene promoter region [132,133]. At the genome-wide level, CHH hypermethylation was observed in developing apple fruits compared to leaves, and comparisons with transgenic fruits also showed an association between lower DNA methylation levels and smaller fruit size [134].

5. The Role of Plant DNA Methylation in Abiotic Stress

5.1. High Temperature Stress

High temperatures and drought are critical climate stressors that significantly impair global crop yields. Projections suggest that global temperatures may rise by approximately 4–5 °C by the end of the 21st century [135]. Prolonged heat stress coupled with insufficient rainfall often precipitates drought conditions. The synergistic effects of heat and drought typically result in more severe damage to crop production than either stressor alone [136]. Both environmental stresses have been shown to induce alterations in DNA methylation, including hypermethylation and demethylation, in both coding and intergenic regions. In response to heat stress, the heat-resistant rapeseed (Brassica napus) genotype Huyou2 exhibited predominantly hypomethylated DNA, in contrast to the more heat-sensitive genotype Fengyou1 [137]. Additionally, in the drought-tolerant crop millet (Setaria italica), methylation of CHG and CHH motifs in the upstream and coding regions of heat shock protein (HSP) genes was enhanced under environmental stress conditions. In heat-tolerant varieties, heat stress-induced hypomethylation of many SiHSP genes in CHG and CHH motifs, which in turn enhanced their expression [138]. A MethylRAD library constructed from heat-treated maize inbred line B73 identified 325 differentially methylated genes (DMGs) [139]. Notably, DMGs associated with RNA splicing exhibited low methylation levels and were highly expressed, suggesting that spliceosome activity is augmented under heat stress. A nucleoproteome analysis of Pinus radiata subjected to high-temperature stress revealed a decrease in the abundance of S-adenosylmethionine (SAM) synthase and S-adenosyl-L-homocysteine hydrolase (SAHH), resulting in altered DNA methylation profiles [140]. SAM is a crucial cofactor and methyl group donor, while SAHH is essential for the regeneration of SAM during methylation-mediated gene silencing. Moreover, it was observed that DNA methylation regulates the activity of plant transit factors under heat stress [140].

5.2. Drought Stress

Drought stress typically induces dynamic alterations in DNA methylation across the plant genome, which are negatively correlated with gene expression [141]. These modifications encompass methylation changes in promoter regions, genomic sequences, and TEs regions. For instance, research on Polygonum persicaria has demonstrated that drought-treated parents enhance the ability of their offspring to develop deeper and faster-growing roots in arid environments by altering their DNA methylation patterns. This cross-generational stress memory suggests that DNA methylation serves as a molecular link between genetic and environmental signals [142]. Moreover, plant hormone regulation plays a significant role in modulating DNA methylation during drought stress. Studies on poplar have shown that hormone-responsive genes exhibit hypomethylation during stress recovery, which facilitates gene reactivation. Additionally, drought-induced abscisic acid (ABA) signaling pathways are closely associated with DNA methylation modifications [143]. In maize, drought-induced differential methylation was detected via AMP-PCR, with changes predominantly occurring in regions within genes responsible for plant survival functions, such as protein synthesis, DNA repair, and amino acid metabolism [143]. These findings suggest that hormone signaling enhances stress tolerance by regulating dynamic DNA methylation changes. The cumulative impact of multiple drought events reflects stress memory in plants, which is intricately linked to DNA methylation. In alfalfa, a two-stage drought stress regime significantly enhanced drought tolerance compared to a single drought event, a phenomenon associated with methylation remodeling of relevant genes [144]. Stress memory enables plants to respond more efficiently to repeated stress, indicating that DNA methylation underpins long-term adaptation to abiotic stress [144].

5.3. Salt Stress

Salt stress can cause changes in DNA methylation levels across the plant genome. This dynamic change usually involves cytosine methylation in all motifs (CG, CHG, and CHH). Studies have shown that in the initial stage of salt stress, demethylation occurs in the promoter region of many genes, which promotes the expression of related stress-resistance genes [145]. For example, Arabidopsis thaliana demethylates the promoter region of some salt-resistant genes (such as SOS1) under salt stress, thereby enhancing their expression levels and improving the plant’s ion elimination ability. On the other hand, the level of methylation in genomic regions is often increased, which may be related to gene silencing and maintenance of genomic stability [145]. In rice, genome-wide methylation analysis showed that salt stress induced differential methylation of a large number of genes involved in osmoregulation, antioxidant response, and signal transduction [146]. It has also been found that the demethylation of transposition factors induced by salt stress may activate the mobility of these elements, thereby increasing genomic instability [147]. However, through the RdDM pathway, these transposable factors are usually re-silenced to maintain genome integrity. Plant hormones and signaling molecules are closely related to DNA methylation regulation in response to salt stress. Abscisic acid (ABA) is an important signaling molecule in salt stress, and several genes involved in its signal transduction pathway (such as NCED3 and PYR1) exhibit significant changes in methylation levels under stress conditions [148]. For example, demethylation of genes associated with ABA synthesis under salt stress can promote ABA synthesis, thereby enhancing stomatal closure and antioxidant capacity [149]. In addition, the ABA signaling pathway also participates in DNA methylation regulation by regulating transcription factors (such as ABI5), forming a positive feedback mechanism. Studies have also shown that there is an interaction between methylation regulation and ion homeostasis maintenance [145,149]. For example, some genes in the SOS signaling pathway are regulated by methylation states that help maintain intracellular Na/K ion balance under salt stress.

The epigenetic memory function of plants enables them to show higher resilience when subjected to repeated salt stress. Studies have shown that DNA methylation is one of the important molecular bases of salt stress memory. For example, in Arabidopsis thaliana, gene demethylation induced by initial salt stress can be partially left over after stress relief and rapidly activate related gene expression during subsequent stress [142,144,145]. Cross-generational genetic studies have shown that salt stress can transmit salt-resistant characteristics to the next generation by affecting the methylation patterns of gametes and embryos [146]. For example, in barley and rice, the offspring of salt-stressed parents showed greater salt tolerance, which was closely associated with changes in methylation patterns of some salt-resistant genes. The function of many salt-resistant genes is directly regulated by DNA methylation [149]. For example, the level of methylation in the promoter region of the OsDREB2A gene, which is involved in osmoregulation in rice, is reduced under salt stress, promoting gene expression. This demethylation may be achieved by activating binding sites of transcription factors such as MYB or bZIP. In addition, genes encoding antioxidant enzymes (such as Superoxide dismutase, SOD, and Catalase, CAT) in plants are also regulated by DNA methylation, and their expression is enhanced by demethylation under salt stress, thus alleviating stress-induced oxidative damage [150]. In addition, the methylation regulation of transposable factors and their adjacent genes also plays an important role in salinity resistance. The increased activity of transposable factors under salt stress may provide the potential for resistance to mutation by influencing the expression of neighboring genes. However, this instability is usually inhibited by the RdDM pathway to avoid unwanted genomic disturbances [151].

6. The Role of Plant DNA Methylation in Biological Stress

6.1. Dynamic Changes of DNA Methylation and Response to Biotic Stress

In plants, DNA methylation patterns within the genome exhibit dynamic changes in response to biotic stress. The methylation levels at CG, CHG, and CHH sites can be rapidly reconfigured to meet stress-related demands. For instance, infection of Arabidopsis thaliana with Plasmodiophora brassicae induces genome-wide DNA demethylation, thereby promoting the expression of genes involved in resistance responses [152]. Specifically, demethylation of promoter regions of genes associated with the biosynthesis and signaling of defense hormones, such as salicylic acid (SA), enhances plant immune responses. Similarly, in rice (Oryza sativa) infected by Xanthomonas campestris, promoter demethylation of numerous defense-related genes facilitates the expression of disease-resistance proteins. Insect herbivory also drives the reprogramming of DNA methylation patterns [153]. For example, in tomato plants (Solanum lycopersicum) subjected to bollworm (Helicoverpa armigera) feeding, demethylation of promoters of insect-responsive genes, including those encoding protease inhibitors, enhances the synthesis of insect-resistance proteins, thereby strengthening the plant’s defense against herbivory [154,155,156,157,158].

Recent advancements in DNA methylation analysis and extensive research on Arabidopsis thaliana mutants deficient in various stages of DNA methylation, maintenance, and demethylation have significantly expanded our understanding of how these processes regulate plant responses to fungal infections [159] (

Table 1. The defense responses of the Arabidopsis mutants with hypomethylated and hypermethylated DNA to different bacterial, viral, and fungal pathogens.

Arabidopsis Mutants	Pathogen	Phenotype	Defense Response	References
DNA Hypomethylation
drd1	Pseudomonas syringae pv. tomato DC3000 (Pst)	Resistant	Enhancement of SA-dependent defense	Dowen et al. [152]
	Plectosphaerella cucumerina	Susceptible	Suppression of JA-dependent defense	López et al. [154]
ago4	Botrytis cinerea	Susceptible	Suppression of JA-dependent defense	López et al. [154]
rdr2	Plectosphaerella cucumerina	Susceptible	Suppression of JA-dependent defense	López et al. [154]
	Pst	Resistant	Enhancement of SA-dependent defense	Dowen et al. [152]
rdr6	Botrytis cinerea	Susceptible	Loss of transfer siRNAs that target pathogen genes	Cai et al. [146]
	Pst	Resistant	−	Dowen et al. [152]
nrpd1	Pst	Resistant	Enhancement of SA-dependent defense	Dowen et al. [152]
nrpe1	Plectosphaerella cucumerina	Susceptible	Suppression of JA-dependent defense	López et al. [154]
	Botrytis cinerea	Susceptible	Suppression of JA-dependent defense	López et al. [154]
	Pst	Resistant	Enhancement of SA-dependent defense	López et al. [154]
nrpd2	Plectosphaerella cucumerina	Susceptible	Suppression of JA-dependent defense	López et al. [154]
	Botrytis cinerea	Susceptible	Suppression of JA-dependent defense	López et al. [154]
	Pst	Resistant	Enhancement of SA-dependent defense	López et al. [154]
nrpd1/nrpe1	Plectosphaerella cucumerina	Susceptible	Suppression of JA-dependent defense	López et al. [154]
	Pst	Resistant	Enhancement of SA-dependent defense	López et al. [154]
drm1/drm2	Plectosphaerella cucumerina	Susceptible	Suppression of JA-dependent defense	López et al. [154]
	Pst	Resistant	Enhancement of SA-dependent defensePrimed state of defense response	Yu et al. [142]
	Cabbage leaf curl virus	Susceptible	−	Raja et al. [80]
	Beet curly top virus	Susceptible	−	Raja et al. [80]
drm1/drm2/cmt3 (ddc)	Agrobacterium tumefaciens	Susceptible	Enhancement of ABA-dependent response	Gohlke et al. [156]
	Pst	Resistant	Enhancement of SA-dependent defense	Dowen et al. [152]
dcl2/3/4	Botrytis cinerea	Susceptible	Loss of siRNAs that move into fungal cells and suppress virulence genes	Cai et al. [146]
	Pst	Resistant	Enhancement of SA-dependent defense	Dowen et al. [152]
	Cabbage leaf curl virus	Susceptible	−	Raja et al. [80]
	Beet curly top virus	Susceptible	−	Raja et al. [80]
DNA Hypermethylation
ros1	Pst	Susceptible	Methylation at the promoter of RMG1 and RLP43	Yu et al. [142]
ros1/dml2/dml3 (rdd)	Fusarium oxysporum	Susceptible	Suppression of defense-related genes	Le et al. [160]

). For example, the triple DNA demethylase mutant rdd (ros1dml2dml3) in A. thaliana, which exhibits high levels of DNA methylation, shows significantly increased susceptibility to Fusarium oxysporum [160]. In this mutant, many downregulated genes are involved in defense-related functions, and their promoter regions are enriched in transposable elements (TEs). Effective resistance to F. oxysporum relies on DNA demethylase activity, which targets TE sequences within defense gene promoters, thereby boosting gene expression.

Interestingly, studies of Arabidopsis mutants impaired in various stages of the RNA-directed DNA methylation (RdDM) pathway have shown contrasting effects on defense responses against biotrophic and necrotrophic pathogens. In general, RdDM-deficient mutants are more sensitive to necrotrophic pathogens but exhibit enhanced resistance to biotrophic ones. For instance, hypomethylated Arabidopsis mutants demonstrate increased susceptibility to the necrotrophic pathogen Botrytis cinerea and the bacterial pathogen Plectosphaerella cucumerina [161]. Mutants like rdr6 and dcl2/3/4 are particularly vulnerable to B. cinerea due to impaired small interfering RNA (siRNA) synthesis, which is essential for silencing fungal virulence genes [146]. In contrast, knockout of RdDM components such as DRM2 homologs in the wheat ancestor Aegilops tauschii leads to enhanced resistance against wheat powdery mildew caused by the biotrophic pathogen Blumeria graminis f. sp. tritici. Furthermore, the Arabidopsis trimutant ddc (drm1drm2cmt3), which lacks proper methylation maintenance, exhibits extreme sensitivity to the necrotrophic fungus Alternaria brassicicola [157,158,159].

6.2. DNA Methylation Regulation of Disease Resistance Genes

Resistance (R) genes are central to plant defense against pathogen invasion. DNA methylation plays a pivotal role in modulating plant resistance by regulating the expression of these genes. Under normal growth conditions, most R genes remain in a methylation-mediated silenced state, thereby conserving energy and minimizing the risk of autoimmune responses caused by their overexpression. For instance, in Arabidopsis thaliana, hypermethylation of the SUPPRESSOR OF npr1-1 CONSTITUTIVE 1 (SNC1) gene maintains its transcriptional silence [155]. However, during pathogen infection, DNA demethylation reactivates SNC1, bolstering the immune response. In addition to R gene regulation, TEs in the vicinity of these genes are often controlled by DNA methylation. Pathogen infection can suppress TE activity via the RdDM pathway, thereby preventing their insertion into R genes and the subsequent disruption of gene function. For example, in rice (Oryza sativa), many TEs located near R genes exhibit significantly increased methylation levels following infection with Magnaporthe oryzae (the causal agent of rice blast disease), contributing to genomic stability [152,155].

6.3. Synergistic Effect of DNA Methylation with Plant Hormone Signaling

Plant hormones play pivotal roles in mediating plant responses to biotic stress, with DNA methylation forming an intricate network that modulates plant defense mechanisms by regulating the expression of genes involved in hormone biosynthesis and signal transduction. Salicylic acid (SA), a crucial signaling molecule in plant immunity, undergoes significant alterations in the methylation status of genes involved in its biosynthesis and signaling in response to biotic stress. For instance, the demethylation of ICS1 (a gene encoding an SA biosynthetic enzyme) and NPR1 (a central regulator of SA signaling) in Arabidopsis thaliana facilitates SA-mediated systemic acquired resistance (SAR) [154]. Jasmonic acid (JA), predominantly associated with defense against herbivores and necrotrophic pathogens, is similarly regulated. Demethylation of key genes in the JA signaling pathway, such as COI1 and MYC2, has been shown to activate these pathways following the herbivore attack, thereby bolstering plant defenses. Ethylene also plays a critical role in coordinating broad-spectrum plant defenses against biotic stress. In rice (Oryza sativa) challenged by blast fungus, significant reductions in methylation levels within the promoter regions of ethylene signaling genes, such as ERF1, enhance the expression of defense-related genes. Conversely, the role of abscisic acid (ABA) in biotic stress responses appears more nuanced, with several genes in the ABA signaling pathway undergoing adaptive regulation via DNA methylation [150,152,157].

7. Prospects of DNA Methylation in Crop Breeding

Natural variations in DNA methylation are prevalent in plants and present significant opportunities for crop improvement. With advancements in gene-editing technologies, these opportunities are increasingly becoming achievable. Numerous instances of natural epigenetic variation have been identified in plants. One well-known example is the radially symmetrical flower mutant of Linaria vulgaris, first described by Linnaeus, which is caused by a hypermethylated allele of the transcription factor CYCLOIDEA [158]. In Arabidopsis, studies on epigenetic variation have revealed essential mechanisms involved in regulating DNA methylation [159]. Beyond model organisms, epialleles in various crops have been shown to control important agronomic traits, such as plant architecture in rice [160], fruit ripening in tomatoes [161], photoperiodic sensitivity in cotton [162], and grain quality in maize [163]. Moreover, DNA methylation plays a role in hybridization dynamics in Arabidopsis [164,165].

Beyond its impact on qualitative traits, DNA methylation variation is also thought to play a significant role in shaping quantitative traits. Epigenetic variations that occur naturally are believed to contribute to the phenomenon of “missing heritability” observed in both animal and plant studies [166]. In Arabidopsis thaliana, variations in DNA methylation are closely associated with environmental factors, particularly in genes related to immunity [167]. Furthermore, scans for positive selection have revealed methylated CG sites (mCGs) in the promoter regions of genes involved in the production of specific metabolites, suggesting that CG methylation may play a role in the adaptive evolution of Arabidopsis [168].

Comparative methylation analyses in crops like maize [169,170], soybean [171], rice [172], and wheat [173] have identified a broad array of differentially methylated regions (DMRs) across diverse population varieties, with some regions linked to domestication or environmental adaptation. Genome-wide association studies (GWAS) of DNA methylation profiles in 263 maize inbred lines revealed that over 60% of these DMRs did not overlap with single nucleotide polymorphisms (SNPs). These studies also found DMRs associated with 156 metabolic traits, suggesting that variations in DNA methylation, rather than traditional genetic differences, may play a crucial role in regulating these traits [168]. While genetic variation can influence DNA methylation patterns [174,175], current research indicates that the impact of DNA methylation on phenotypic variation may be underappreciated and requires further exploration. In tomato and maize, hybridization-induced epialleles behave similarly to somatic mutations, with changes that can persist through several generations of backcrossing and self-pollination [156,176,177]. This suggests that epigenetic modifications resulting from hybridization could contribute to phenotypic diversity in traditional breeding programs. Furthermore, epigenetic recombinant inbred lines (epiRILs) provide additional support for the role of epigenetic variation in crop improvement. In Arabidopsis, epiRILs exhibit pleiotropic phenotypic variation, some of which is comparable to the phenotypic differences observed in recombinant inbred lines (RILs) [178,179]. Likewise, artificial selection of traits such as energy efficiency in genetically uniform populations of rapeseed and rice has produced epigenetic lines that significantly outperform conventional varieties in the field, highlighting the practical value of epigenetic selection for enhancing crop yields [180,181,182].

The introduction of epigenetic variation into traditional breeding programs is often constrained by extended breeding cycles and the phenomenon of epigenetic drag, wherein undesirable epigenetic mutations are inadvertently propagated. When specific target genes are identified, epigenome editing emerges as a precise and controllable strategy for enhancing agronomic traits. Numerous epigenome editing tools have been developed in plants, facilitating the study of DNA methylation regulation [183]. These tools generally comprise a targeting module that directs proteins to specific genomic loci, a modification module that alters epigenetic states, and, optionally, an auxiliary module to modulate the activity of the modification process.

Among the various epigenetic editing systems, CRISPR-dCas9 has gained significant attention due to its ease of design and construction, particularly when compared to traditional systems such as TALE and artificial zinc finger proteins [165]. In addition to the standard use of dCas9, the nuclease activity of Cas9 can be inhibited by shortening the spacer region of guide RNAs [184]. The modifier domains, responsible for inducing epigenetic changes, are highly versatile. These range from the catalytic domain of the DRM gene to prokaryotic DNA methyltransferase MQ1, mammalian DNA demethylase TET1, and components of the RNA-directed DNA methylation (RdDM) pathway, all of which have been successfully applied for DNA methylation editing in Arabidopsis [62,185,186,187]. Advanced systems like SunTag and MS2 can be integrated into the targeting module to increase the local concentration of effector domains. Although still in the early stages of development, precise epigenome editing shows great potential for efficiently modulating the epigenetic states of specific genes in crops. However, realizing its full potential in breeding programs requires addressing several challenges: minimizing off-target effects, improving the cross-generational stability of edits, and accurately predicting optimal guide RNA target sites. In mammalian cells lacking DNMT activity, the dCas9-Dnmt3a modifier exhibits widespread activity, although ectopic methylation primarily occurs at sites that were already methylated, with minimal impact on gene expression [188]. Off-target effects can be reduced by lowering overall effector levels while enhancing their local concentration [189]. These findings highlight the need for more precise regulation of DNA methyltransferase or demethylase activity, as compared to the endonuclease activity of Cas9. Moreover, targeted de novo methylation of thousands of promoters in human cells has shown environmentally contingent stability and transcriptional responses [190]. Given that chromatin state maintenance relies on self-reinforcing feedback loops involving multiple components, disrupting these loops and creating new ones may necessitate simultaneous editing of several epigenetic marks [191]. Achieving precise transcriptional regulation of target loci also requires a thorough understanding of their cis-regulatory elements and trans-regulatory factors.

8. Conclusions and Prospect

The escalating frequency and severity of extreme weather events driven by global climate change pose mounting challenges to agricultural production. Stresses such as elevated temperatures, drought, salinization, and pest infestations critically undermine crop yield and quality, highlighting the pressing need to develop crop varieties with enhanced adaptability and robust stress resistance. Recent advances have unveiled the pivotal role of DNA methylation in mediating plant responses to environmental stresses. Its inherent plasticity and heritable characteristics offer a promising theoretical foundation and a valuable tool for advancing crop breeding strategies.

DNA methylation plays a pivotal role in regulating plant responses to environmental stress and disease resistance by modulating gene expression. Leveraging this mechanism, modern molecular breeding techniques can be employed to enhance crop resilience through targeted adjustments of methylation patterns in specific genes. Traditional agriculture heavily relies on fertilizers and pesticides to sustain high yields, a practice that significantly pollutes the environment and poses risks to food safety. Investigations into the role of DNA methylation in nutrient uptake and stress responses have revealed its potential to enhance nutrient use efficiency in crops, thereby reducing reliance on chemical inputs. For instance, under nitrogen-deficient conditions, certain plants improve nitrogen absorption by modifying the methylation status of key genes, offering a novel strategy for breeding nutrient-efficient crop varieties. Moreover, regulating DNA methylation can bolster the natural disease resistance of crops, thereby decreasing pesticide requirements. This dual benefit—sustaining high yields while mitigating environmental harm—positions DNA methylation as a cornerstone in the development of sustainable green agriculture. Recent advancements in genomics, transcriptomics, and epigenetics have further enriched our understanding of crop trait improvement. By integrating multi-omics datasets, researchers can unravel the complex roles of DNA methylation in gene regulatory networks with greater precision. For example, analyses of stress-resistance traits frequently link dynamic methylation changes to the upregulation or downregulation of specific genes. Multi-omics approaches thus enable the identification of key regulatory genes and pathways, providing a robust scientific foundation for precision breeding.

The dynamic and multifaceted nature of DNA methylation offers immense potential for advancing crop breeding, yet it also presents significant challenges. Environmental factors influence DNA methylation patterns, raising critical questions about their stability and heritability that warrant further investigation. Moreover, the regulatory mechanisms of DNA methylation vary across species and genomic contexts, necessitating careful consideration when developing universal breeding strategies. The precision and safety of epigenetic editing technologies likewise require further refinement to enable their broad application in agriculture. Looking ahead, deeper insights into the role of DNA methylation in plant growth, development, and stress responses, combined with cutting-edge approaches such as genome and epigenetic editing, could revolutionize the breeding of high-yield, high-quality, and stress-resilient crops. International collaboration and interdisciplinary integration will be crucial for building comprehensive epigenetic data repositories spanning diverse crop species, thereby providing robust datasets and technical resources to advance breeding efforts.

In conclusion, harnessing DNA methylation in crop breeding has the potential to address pressing challenges in global agriculture while fostering the development of efficient, sustainable, and environmentally friendly farming practices. This field of research is poised to drive a transformative revolution in crop breeding, enhancing global food security and agricultural productivity.