DNA methylation in wheat: current understanding and future potential for enhancing biotic and abiotic stress tolerance

Abstract

DNA methylation is a paramount epigenetic mark that helps balance gene expression post-transcriptionally. Its effect on specific genes determines the plant’s holistic development and acclimatization during adversities. Triticum aestivum L., an allohexaploid, is a dominant cereal crop with a large genome size. Changing environmental conditions exert a profound impact on its overall yield. Here, bibliometric science mapping was employed for a nuanced understanding of the prevailing research trends in the DNA methylation study of wheat. The detailed data obtained was used to delve deep into its fundamentals, patterns and mechanism of action, to accumulate evidence of the role of DNA methylation in the regulation of gene expressions across its entire genome. This review encapsulates the methylation/demethylation players in wheat during different stages of development. It also uncloaks the differential methylation dynamics while encountering biotic and abiotic constraints, focusing on the critical function it plays in fostering immunity. The study significantly contributes to broadening our knowledge of the regulatory mechanism and plasticity of DNA methylation in wheat. It also uncovers its potential role in improving breeding programs to produce more resilient wheat varieties, stimulating further research and development in the field.

Supplementary Information

The online version contains supplementary material available at 10.1007/s12298-024-01539-1.

Introduction

Triticum aestivum L. (Bread wheat), belonging to the family of Poaceae, is a dominant cereal crop across the globe, having an enormous genome size of 17 Gb (Consortium et al. 2018). It is a self-pollinating hexaploid with cleistogamous flowers and constitutes of three highly similar sub-genomes, AA, BB, and DD (~ 5.5 Gb each), derived from Triticum durum (AABB) and Aegilops tauschii, (DD). Like any other plant, wheat also lacks the established immune system as in animals, yet it has stunningly evolved defense mechanisms that act when encountering any stimulation (Zhao et al. 2024). Environmental conditions govern wheat varieties’ geographical distribution, prolificness, growth, and development (Rodrigues et al. 2024). They require optimum conditions for survival and proliferation, which could limit their growth and, in turn, the overall yield. When the crop experiences a state of stress (biotic and abiotic), it causes damage to the genomic integrity of the plant. Wheat yield loss due to increased temperature and drought conditions is estimated to be around 5.5–12% (Zulkiffal et al. 2021). Every 2 °C increase in temperature is detrimental to the total turnover (Challinor et al. 2014). Pathogen invasion is another dominant factor contributing to the dwindling annual harvest. Predominantly, rust pathogens attack the most, resulting in approximately 15–20% yield loss per year (Poudel and Kc 2024).

Classical genetics cannot uncover and explain the genotypic variations among them with the varying environmental and geographic conditions. Here, the mechanism of epigenetics helps to solve some enigmas. When the constitutive defense of a plant, i.e., its preformed barrier, is compromised, its inducible defenses along with genetic rearrangements and epigenetic modifications are enforced. This might be inherited with the genome generations after generations, resulting in the altered morphology (phenotypic characteristics) and signalling pathways, to overcome the perturbations of the environment. Epigenetics refers to the changes in gene expression other than any alterations in the DNA sequence (Boyko and Kovalchuk 2007). The various epigenetic signalling tools include noncoding RNAs (ncRNAs) mechanisms, X-chromosome inactivation, genomic imprinting, paramutation, chromatin remodeling, histone modification, DNA methylation, etc. Among the main methods of epigenetic modifications in plants, DNA methylation is a dominant player (Aboud et al. 2021). Crop improvement initiatives have been strengthened by recent developments in our understanding of how epigenetic variations, such as DNA methylation, affects the phenotypic traits of plants. Crops are being improved rapidly by precisely altering their nucleotide sequences through the use of genome editing technologies, especially CRISPR-Cas9. In addition to these genetic alterations, epigenetic modifications provide the possibility of overcoming genome editing’s drawbacks, including off-target effects and gene knockout, which can only cause the loss of function of particular genes (Venezia and Krainer 2021; Ueda et al. 2023).

Agronomically significant features like flowering time, seed dormancy, and yield are largely regulated by DNA methylation. Epigenetic biomarkers provide a distinct benefit in crop breeding because, in contrast to genetic variations, they can pass on environmentally adapted traits to offspring. Crop stress resistance mechanisms, such as wheat’s ability to withstand salt and the ability of pea and barley to withstand drought, have all been linked to DNA hypermethylation (Liu and He 2020; Sun et al. 2022; Yin et al. 2024). On the other hand, responses to nitrogen fixation and heat stress in soybeans and rapeseed, drought and salt stress in rice and faba beans have been linked to DNA hypomethylation, indicating its function in transferring advantageous heritable modifications that support crop domestication and evolution (Yang et al. 2020; Wang et al. 2021; Jianing et al. 2022; Xun et al. 2024).

A targeted DNA-binding domain and a functional domain are commonly found in site-specific epigenome editing tools, which alter the epigenome at certain loci. Zinc Finger (ZF) proteins, Transcription Activator-Like Effectors (TALEs), or Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR-Cas9) can serve as the basis for the targeting domain (Waryah et al. 2018). It has been demonstrated that ZF proteins, which are naturally occurring DNA-binding motifs, can effectively induce DNA methylation in Arabidopsis thaliana to control flowering time when combined with RNA-directed DNA methylation (RdDM) (Miglani et al. 2019). The DNA demethylase, Ten-eleven translocation (TET1) has been used in ZF-based epigenome editing to induce demethylation; however, the demethylation impact may be temporary since RdDM machinery may revert back to its fully methylated state (Qi et al. 2023). Despite being more expensive and labor-intensive than ZFs, TALEs provide greater specificity and design flexibility that enable the recognition of individual DNA bases. Certain applications have been deemed superior to CRISPR-Cas9 and have shown effectiveness in modifying gene expression (Cheng et al. 2024). The application of CRISPR-Cas9 for site-specific epigenetic alterations has been made possible by recent advancements. While fusion with the TET1 catalytic domain can activate genes by increasing DNA demethylation, CRISPR-Cas9 can induce gene silence through DNA methylation when fused to DNA methyltransferases. As an example, the FLOWERING WAGENINGEN (FWA) promoter was effectively methylated in Arabidopsis thaliana using CRISPR-Cas9, resulting in an early blooming phenotype (Ghosal et al. 2021).

Innovations and new understandings are important to finding newer approaches without duplicating the same problems. In the past few years, bibliometric indicators have been frequently used to analyze the progress in sustainable and reliable agronomy (Awhari et al. 2024). Exploring the scientometrics references, combined with data interpretation, gives the researchers a cognizance of the data inside a field. A total layout of the experiments performed to date can be made by depicting the characteristics and qualities of the information provided.

This review offers exploratory information regarding the current state of DNA methylation studies in wheat. The bibliometric analysis identifies the emerging research topics and changing trends in this field uncloaking the potential research directions for future research. It elaborates on the DNA methylation patterns in wheat during different developmental stages, with particular attention to its plasticity during environmental constraints. Crop improvement strategies that could improve wheat varieties’ traits have also been discussed. The study would support more research on the heritable epigenetic variation resulting in phenotypic characterization in wheat, which can produce much healthier and broad-spectrum resistant varieties.

DNA methylation: hidden driver of plant growth

In plants, RNA directed DNA methylation (RdDM) pattern is observed where the use of small interfering RNAs or siRNAs (24 nucleotides in length) is significant. It was first discovered in transgenic potatoes with viroid genes (Wassenegger et al. 1994). The RdDM pathway consists of the siRNA biogenesis pathway followed by de novo DNA methylation using two RNA polymerases, Pol IV and Pol V (Fig. 1a), respectively and eventually favour Post Transcriptional Gene Silencing (PTGS) by collaborating with a chromatin remodeler, SWI/SNF and thereby facilitating alteration in the nucleosome positioning (Li et al. 2006). Unlike cytosine methylation (the dominant form of methylation in plants), N6-Adenine methylation (6-mA) is typically considered an activator of transcription. DNA adenine methyltransferase (DAMT) makes explicit methylation marks. Oxidation occurs at the methyl group of 6-mA and through different pathways methylation and demethylation occur in a roundabout way (Fig. 2a) (Sedgwick et al. 2007).

Fig. 1.

a RNA directed DNA methylation (RdDM) consists of siRNA biogenesis followed by de novo DNA methylation. SAWADEE HOMEODOMAIN HOMOLOGUE 1 (SHH1) directs RNA polymerase Pol IV to the target loci. DNA-dependent Pol IV with its subunit NUCLEAR RNA POLYMERASE D1 (NRPD1) synthesizes a single-stranded RNA (ssRNA), which later converts to double-stranded RNA (dsRNA) with RNA-dependent RNA polymerase (RDR2). Chromatin remodeller CLASSY1 (CLSY1) facilitates the passage of Pol IV along the chromosomal locus. The dsRNA gives rise to 24 nt siRNA after cleavage by Dicer-like protein (DCL3), which then methylates their 3ˈOH ends to stabilize the molecule by HUA ENHANCER 1 (HEN1). These are then loaded onto ARGONAUTE 4 (AGO4) where it binds to the noncoding RNAs (ncRNA; scaffold RNAs) which are RNA polymerase, Pol V transcripts. The KOW DOMAIN-CONTAINING TRANSCRIPTION FACTOR 1 (KTF1) communicates with the biggest subunit of Pol V, NUCLEAR RNA POLYMERASE E1 (NRPE1) and volunteers AGO4 to the latter’s carboxy-terminal domain (CTD). Here, AGO4-bound siRNA base pairs with the nascent Pol V transcript and forms the functional RdDM effector complex by recruiting (DNMT3) family DNA methyltransferase: DOMAINS REARRANGED METHYLTRANSFERASE 2 (DRM2) to catalyze de novo DNA methylation. dsRNA binding protein INVOLVED IN DE NOVO 2 (IDN2) alongside its two partially redundant paralogues, IDP1 and IDP2 collaborates with chromatin remodeller SWI/SNF to stabilize base-pairing among siRNAs and the Pol V transcript and favour Post Transcriptional Gene Silencing (PTGS). b Represents DNA demethylation in plants

Fig. 2.

a Reversible adenine methylation. The DAMT-1 transfers the methyl group from S- adenosyl methionine (SAM) to the exocyclic NH2 on the purine ring at the 6th position (6-mA) which returns back to the unmodified state by the action of AlkB oxidase by the oxidation of N6 methyladenine into intermediate products, N6-hydroxymethyladenine and N6- formyladenine consecutively, which later releases formaldehyde and converts back to adenine. In another pathway, hypoxanthin is produced as an intermediate product by the deamination process of 6-mA deaminase, which later enters BER as hypoxanthin, which is then broken down by glycosylase to produce an abasic site, which is then rectified by endonuclease, DNA polymerase and ligase activity. Cyclic N at the N1 position gets methylated, encountering an alkylating agent, which reverts back to its original adenine state by the action of AlkB oxidase. b Cytosine demethylation. The modified deoxycytidine, i.e., methyl cytosine, by the action of a ten-eleven translocation enzyme, gets converted to intermediate products like hydroxyl methyl deoxycytidine, formyl deoxycytidine and carboxy deoxycytidine consecutively. Thymine DNA glycosylase (TDG) belongs to the uracil DNA glycosylase family and works only on formyl and carboxy deoxycytidine. It forms an abasic site which later is repaired by BER to return it to the unmodified state

While 5-mC is believed to increase the DNA helix stability, the helical design of DNA weakens by 6-mA. In this way, 5-mC, when occurring in the promoter region, is viewed as a repressor of the transcription process and 6-mA is believed to function as an activator of transcription. In Arabidopsis, the methylation catalysis of cytosine and adenine is achieved by DRM2, which contrasts with wheat, where N6-adenine DNA-methyltransferase was accounted for to play out this methylation process. Active and passive DNA (de)methylation monitors the methylation throughout the cellular processes. Conceptually all the known bases can be modified, but adenine and cytosine methylation has been affirmed to date. Apart from methylated cytosine (5-mC) and N6-methyladenine (6-mA), other products such as 5-hydroxymethylcytosine (5-hmC), 5-formyl cytosine (5-fC), 5-carboxyl cytosine (5-caC), has also been identified (Klungland and Robertson 2017) but cytosine methylation remains a distinguished epigenetic mark in plants. Though not a very significant amount of evidence supports the presence of 5-hmC, some reports still propose its presence in plants, however, with clashing derivations (Mahmood and Dunwell 2019). In an article, the appearance of 5-hmC in A. thaliana and some other plant species was affirmed but in a significantly smaller amount, concluding that it might not be available in a naturally appropriate amount in the plant genome (Erdmann et al. 2015). A meagre amount of 5-hmC was detected (1.39–2.17 per million nucleosides) as per studies in three rice cultivars (Wang et al. 2015).

Cytosine demethylation involves the enzymatic conversion of 5-mC with the intermediate product formations (Fig. 2b) like 5-hmC, 5-fC, and 5-caC, which BER accompanies. Widman et al. (2009) have highlighted how epigenetic reprogramming for overcoming environmental constraints and developmental processes requires the activation of specific genes, which is aided by DNA demethylation in plants. DNA glycosylases create an abasic site (Fig. 1b) in the 5′ and 3′ end of the gene and not the gene body by eliminating 5-mC where later BER takes place without any active replication and the gap is filled preventing hypermethylation. However, this pathway cannot be the only method to achieve demethylation as it would destabilize the genetic integrity by producing numerous abasic sites and broken DNA strands. Although the TET enzyme is not present in plants, the human TET has been shown to cause epimutagenesis in Arabidopsis and intermediate products have also been found in plants (Mahmood and Dunwell 2019). The feedback mechanism is observed between the RdDM and the demethylation mechanism as a mutation caused to any of the components of RdDM results in a drastic decrease in ROS1 transcript level (Mathieu et al. 2007). It has also been observed that the mutation of RdDM results in a decreased level of demethylation occurring by ROS1 to protect the genome from excessive methylation (Mosher et al. 2008).

Data acquisition for crafting insight into DNA methylation in wheat

Data from wheat research for the DNA methylation category, available in the authoritative and reliable academic database, Web of Science (WoS) core collection (https://www.webofscience.com/wos/woscc/) (1989-present) was surveyed. In the methodology, “Article title, Keywords, Abstract, Organization, Nationality, Total cited reference count and Publication type” were used. The search was carried out in the keyword query with wheat and DNA methylation. Full records and cited references were extracted for bibliometric analysis. Network, overlay and density visualization of the obtained metadata was done through VoS viewer program (https://www.vosviewer.com/). Patents were also checked through the Patent search tool (https://iprsearch.ipindia.gov.in/publicsearch) to explore the trends of patents with the above-mentioned keywords. The same data with reviews and original research articles assisted in summarizing the methylation mechanism in wheat with their identified key players unveiled to date during its overall growth and development.

Bibliometric overview of DNA methylation research in wheat

The selection of publications with wheat and DNA methylation as co-occurring words offer specialization on specific literature to capture conceptually rich information (Fig. 3). Collaborations, impact factors, and author’s affiliations were not emphasized upon in this review, in order to focus mainly on the type of trending research in this field and the substantial surge in the number of publications related to the involvement of DNA methylation in assisting growth and development of the crop under varying parameters. Based on the understanding of DNA methylation in plants and related articles, its mechanism of action in wheat has been summarized in the upcoming sections.

Fig. 3.

a Network visualization map of keyword wheat; DNA methylation co-occurrence separated into 6 clusters of different research topics ranging from evolution (dark blue), wheat genome (green), DNA methylation (red), epigenetics in other plants (purple), epigenomic mutations (light blue) and epigenetic regulation and stress tolerance (green). b Density visualization map of the trending research areas of wheat and DNA methylation showing a major research gap of correlational research among the two keywords with the intensity of research trend highlighted in yellow. c Countries with the highest citations and d top organizations to carry out research in this field

549 articles were analysed from the WoS core collection published to date. For the analysis a keyword’s minimum number of occurrences was set as 4. All keywords used for the particular study were separated into 6 clusters of different research topics ranging from growth and development, biotic and abiotic stress, mechanism of DNA methylation, differential methylation of regulatory genes and evolution. It was observed that the majority of studies associated with this field commenced in 2010. 194 keywords met the threshold and uncovering the role of methylation during different stages of development and environmental constraints emerged as trending research since 2015. The density visualization map remained consistent with the overlay visualization map with very little co-occurrence of the two topics (Fig. 3a and b). It was determined that majority of research conducted with the keywords, “DNA methylation” and “wheat” were focused mainly on differential expression analysis and the effect of DNA methylation on transposable elements, evolution, allopolyploidization and germination. With respect to stress tolerance studies, primarily drought and salt tolerance have been studied with little or no data on biotic stress responses during plant-pathogen interaction.

The analysis of articles and review articles were published majorly from 27 countries, with the highest citations from the USA (5725), the People’s Republic of China (3492) and Germany (1571) (Fig. 3c). The Chinese Academy of Science was found to be the top organization that carried out the greatest amount of research in this field (Fig. 3d). None of the articles of the WoS metadata had any patent. In the patent search engine, 79 patents were recorded with the keyword “DNA methylation” and 163 patents with the keyword “Wheat”. However, validating the results of WoS analysis, no patents were registered with the co-occurrence of these two words with maximum patents registered for the co-occurring keywords for cancer, aromatic compounds, cell lines, animal cells and disease detection. However, some generalized patents primarily for the determination of DNA methylation levels were available (Supplementary Table S1).

Thus, the study shows that although wheat and DNA methylation have both been investigated in great detail, they seldom ever co-occur. The intersection of wheat’s DNA methylation and stress tolerance, in particular, has not received much attention in the literature. This is a significant research void. The epigenetic mechanisms that contribute to wheat’s resilience to environmental stressors, including DNA methylation, are still poorly understood, despite the fact that DNA methylation has been extensively examined in a variety of animals and wheat has been intensively studied for its agronomic properties. The gap proposes a multidisciplinary strategy that integrates genomics, epigenetics, and the physiology of wheat to examine the relationship between DNA methylation patterns and wheat stress tolerance features. In order to ascertain whether DNA methylation is a stable and heritable feature that can improve stress tolerance in wheat, researchers should use a longitudinal strategy to track DNA methylation dynamics over time and under multiple stressors since stress situations frequently overlap in nature. Integrating phenotypic information on stress tolerance with epigenomic information (such as DNA methylation profiles) will be a significant advancement. The development of stress-tolerant wheat cultivars may be aided by the discovery of epigenetic biomarkers that can forecast stress tolerance. An intriguing way to investigate how precise DNA methylation changes can increase wheat’s resistance to environmental stress is through the application of CRISPR/Cas9 technology for targeted epigenetic modifications. By addressing these gaps, future research could make significant contributions to improving wheat’s resilience to climate change and other environmental challenges.

DNA methyltransferases in wheat

In plants, Chromomethylase 3 (CMT3) and MET 1 usually is accountable for maintaining the cytosine methylation in the symmetric CG and CHG (where H can be A, C or T) sites (Law et al. 2010). The asymmetric (CHH) site methylation is kept up with by de novo pathway or by the chromatin remodeler DDM1-dependent chromomethylase 2 (Zemach et al. 2013). Based on the experimental observations in several plants, such as rice (Sharma et al. 2009), maize (Candaele et al. 2014), and Arabidopsis (Huang et al. 2010), it can be summarized that the methyltransferases can be categorized into four distinct classes—Dnmt/MET1 (Methyltransferases1), Dnmt2, CMT (Chromomethyltransferases) and Dnmt3. Out of the four, CMT catalyzes CpNpG methylation and is not present in animals. Interestingly, both CMT and Dnmt1 have a BAH (Bromo-adjacent homology) domain that links DNA methylation to replication (Papa 2001). De novo methylation in mammals and plants is mainly carried out by the Dnmt3 class of enzymes. However, the mechanism of action is different. In plants, DRM2 de novo methylation activity is targeted to siRNAs, which is not the case in mammals. Instead, the PIWI-associated RNA (piRNA) pathway, somewhat similar to the RdDM pathway, guides Dnmt3 activity in mouse germ cells (Law et al. 2010).

In wheat, 53.30% of CpG, 3.48% of CpHpG and 1.41% of CpHpH methylation have been observed (Gahlaut et al. 2020), which is quite similar to the methylation pattern of other plant species where highest methylation is seen in CpG islands and lowest in CpHpH sites.

Not much data on wheat methyltransferases is available. Earlier, the presence of five putative DNA methyltransferase genes was reported, namely, TaMET1, TaMET2a, TaMET2b, TaCMT, and TaMET3 (Dai et al. 2005), where TaMET3 and TaMET2b showed sequences similar to Oryza sativa. At the same time, the rest were identical to the methyltransferases of Zea mays, where TaMET2a contained 8–10 conserved motifs (a characteristic feature of cytosine methyltransferase), which strongly points towards its function as DNA methyltransferase (Thomas et al. 2014). TaMET1 and TaCMT catalyze mCG and CNG methylation, respectively. Also, the presence of TaMET1 and TaMET3 drastically varies according to the dormancy state of wheat seeds. Its high proportion of mRNA present in dry seeds suggests that the dormancy state of the seeds might be related to the methylation status of the genes (Dai et al. 2005). TaMET1, the maintenance methyltransferase of wheat, has been further explored to reveal nine copies of the methyltransferases suggesting that the genomic region where MET1 lies in the hexaploid wheat might have undergone duplication before the hybridization of the ancestor plants, i.e., Triticum durum and Aegilops tauschii, resulting in the organization of TaMET1 in three paralogous groups of chromosomes 2, 5 and 7. Thus, resulting in 9 TaMET1 loci. Interestingly, very recently, 52 methyltransferases have been discovered in wheat (Gahlaut et al. 2020), which showed the presence of numerous methyltransferase genes (Fig. 4) belonging to the category of CMT, MET, DNMT2, and DRMs (Domains rearranged methyltransferases), justifying the notion of duplication of the genes as most of the genes were present in all the sub-genomes. However, not many studies have been done about the characterization of MET in wheat compared to other monocot crops like rice and maize, and in-depth knowledge regarding the evolution of MET in wheat and its categorization during polyploidy is still needed.

Fig. 4.

List of the 52 methyltransferases of three sub-families of DRM-26, CMT-14, MET-9, and DNMT-3 reported in wheat along with the domains present in the different methyltransferases with CHRomatin Organization MOdifier Domain (CHROMO), Bromo Adjacent Homology (BAH) Domain, DNA-Methyltransferase (DNA-Meth), Replication Foci Domain (RFD) and UBiquitin-Associated (UBA) Domain (Gahlaut et al. 2020)

The presence of N6-DNA adenine methyltransferase is not an incidental phenomenon and is essential for the growth of rice and Arabidopsis (Liang et al. 2018). Wheat adenine DNA methyltransferase, wadmtase has been isolated and properties unfolded. However, their discovery is still on hold and the mechanism of action still unclear.

Genome-wide DNA methylation pattern of wheat

It is assumed that region methylation is better than single-position methylation for regulating gene expression. However, the situation may vary. It is also believed that methylation in gene promoters inhibits regulation and affects protein binding resulting in transcriptional repression. In Arabidopsis, mCpG is usually present in coding regions, while CpG, CHG, and CHH methylated sites are seen in noncoding regions (Widman et al. 2009). Unlike Arabidopsis, in wheat, CpG methylated sites are maximum in the transcribed region and CHH/CHG in the non-transcribed region (in a much lower percentage) (Gardiner et al. 2015; Saroha et al. 2024). Also, like other plants, transposons are hypermethylated, probably to reduce their mobilization (Ding et al. 2024). Gardiner et al. (2015) have proposed three methylation patterns, the uni-genome, bi-genome, and tri-genome methylation, where methylation occurs in a single sub-genome, in two sub-genomes or all three sub-genomes, respectively. In the transcriptional region, uni-genome and bi-genome methylation are in the highest proportion in CpG sites rather than in CHH or CHG sites, but in the non-transcribed regions, CpG site methylation is much less as compared to gene body regions. In contrast, the CHG and CGG site methylation percentage increases to that of the gene body region. The same is seen in bi-genome methylation. These observations are in support of the methylation pattern of other plants. Still, the tri-genome methylation in all three sites is highly conserved between the sub-genomes in the transcribed and non-transcribed regions (with specific ratio differences). Thus, the tri-genome methylation is highly conserved, and the sub-genome-specific methylation pattern is variable. The tri-genome methylation is very similar to the Aegilops taushii, which is proposed to be the donor of sub-genome DD. Interestingly, the sub-genome D methylation shows similarity to the progenitor compared to the tri-genome. However, a more detailed study is required in polyploidy genomes such as wheat, where the control on the gene dosage between three sub-genomes could be checked through differential methylation of homoeologous genes.

Differential methylation status of dry and germinating wheat seedlings

Significant differences have been observed in the methylation pattern of embryo and endosperm of germinating and dry seeds of wheat. The DNA methylation accounts for 27% on average in the germinating seeds, as revealed by Methylation Sensitive Amplification Polymorphism (MSAP) profiling, which is considerably higher than rapeseed (15.7%; Lu et al. 2006) and rice (16.3%; Xiong et al. 1999). However, the percentage is much lower than Arabidopsis (35–43%; Cervera et al. 2003). As compared to the germinating wheat seeds, the dry seeds have a much higher methylation percentage, showing that dormant wheat seeds have a higher amount of 5-methylcytosine (Malabarba et al. 2021) than the germinating wheat seeds. During seed germination of wheat, a majority of methylation can be controlled by retrotransposons (Meng et al. 2012).

DNA methylation and stress responses in wheat

Epigenetic processes include many genes in the stress response to stimuli (Wu et al. 2019; Shahid 2019). Therefore, in the last decade, the stress responses of wheat, along with its signalling pathways, have gained popularity in the research world, which focuses on environmental factors and wheat’s response to various constraints by altering DNA methylation (Kumar et al. 2023). It has been seen that both water deficit and change in osmotic pressure have a direct impact on the methylation level of wheat seedlings (Duan et al. 2020). The 5 mC accumulation in the leaf of the wheat seedling is directly proportional to the increasing water deficit highlighting significant tissue and species-specific responses to water deficit. The results show that the methylation patterns in the genomic DNA of wheat seedlings, particularly in the leaf tissue, increased significantly under water deficit conditions. This increase was particularly pronounced in the leaf of wheat AK58, suggesting a more robust response to stress compared to wheat XM13. Complete methylation was the dominant form in the genomic DNA, and under water deficit, the increase in methylation was significantly higher than that of demethylation. The DNA methylation response also exhibited notable polymorphisms, with the root tissue, especially in AK58, displaying higher methylation variability. Such findings can be linked to breeding techniques, such as CRISPR/Cas9 and other epigenomic editing techniques, for improving wheat’s resilience to water deficit to modify the DNA methylation landscape at key regulatory loci involved in stress responses that regulate drought tolerance, potentially enhancing their expression or silencing undesirable pathways. Additionally, the tissue-specific and species-specific methylation changes observed between wheat AK58 and XM13 suggested that DNA methylation patterns can be exploited as markers for breeding. Marker-assisted selection (MAS) could be used to identify wheat lines with desirable methylation patterns associated with water-deficit tolerance. A similar correlation was observed with respect to osmotic pressure which resulted in a higher accumulation of 5mC in the wheat leaf. Also, genome-wide hypomethylation observed during salt stress (Lan et al. 2009), might have a direct impact on the downregulation of the high-affinity sodium channel (HKT1) responsible for maintaining the sodium influx in the plant (Kaur et al. 2018). In addition, it has also been reported that DNA methylation is related to Abscisic acid (ABA) responsiveness (Kim et al. 2019). Since ABA is involved in a plant’s adaptability to abiotic stress, it is necessary to study it in wheat since a clear explanation of its correlation with epigenetics is lacking.

The presence of resistant genes like leaf rust resistance (Lr) gene, Lr28/Lr24 during plant-pathogen interaction alters the methylation status of the wheat plant. Meanwhile, both hypomethylation, as seen in the case of NBS-LRR and JUB1, provides resistance against the pathogen (Gullner et al. 2018; Afreen and Kumar 2024) and hypermethylation results in a negative regulator of DMGs, inferring that these phenomena might lead to silencing of the genes (Saripalli et al. 2019). In our previous study of gene TaJUB1-L (Afreen and Kumar 2024), we examined the methylation states of two small open reading frames (sORFs) located in the 3′ untranslated region (UTR) of the wheat homolog of JUB1, specifically at cytosine residues in CpG, CHH, and CHG contexts. These modifications were assessed at various time points during leaf rust (Puccinia triticina) pathogenesis in two near-isogenic lines of wheat (HD2329), differing in the presence or absence of the leaf rust resistance gene Lr24. The analysis revealed a significant demethylation of CpG dinucleotides within the sORFs of the 3′UTR in the resistant isolines 24 h post-infection. Notably, this demethylation correlated with upregulated expression of TaJUB1-L, indicating that changes in DNA methylation at the 3′ UTR may serve as a regulatory switch for gene expression. Moreover, it was also reported that the dynamic methylation of the CHH site affects the resistance of wheat progenitor Aegilops taushii against the fungal attack of an obligate parasite Bluemeria graminis, which also points towards the active involvement of DNA methylation during pathogen attack. Additionally, MSAP profiling also revealed alteration in the methylation pattern of Lr19 and Lr41 during leaf rust pathogenesis (Fu et al. 2009).

From a breeding perspective, such results offer valuable information for the development of advanced crop improvement strategies, as previously discussed. By precisely targeting epigenetic modifications at specific loci, such as the methylation patterns in the 3′ UTR of TaJUB1-L or other defense-responsive genes, it may be possible to fine-tune the expression of these genes to enhance disease resistance in wheat. Such strategies could be implemented to either activate or repress genes of interest in a controlled manner, potentially improving resistance to pathogens like leaf rust while minimizing off-target effects. Furthermore, the ability to utilize conventional methylation assays such as bisulfite sequencing to monitor and predict gene expression changes in response to pathogen attacks could become an invaluable tool for breeders. These assays could be integrated into marker-assisted breeding programs or used to guide the design of targeted genome-editing approaches, offering a cost-effective and reliable means to fine-tune plant resistance traits.

Wide wheat varieties have been developed in recent years against different stress conditions (Table 1), which points out the important function of DNA methylation in controlling gene expression. However, a specific methylation pattern is not followed to overcome all the constraints. It varies differently with different environmental conditions, making it difficult to conclude a fully established statement about the pattern they follow and the genes they target in general.

Table 1.

Wheat varieties with differential methylation status during biotic and abiotic stresses

Environmental condition	Contrasting wheat variety		Methylation status	References
	Tolerant	Sensitive
Drought	C306	HUW468	Higher demethylation in tolerant variety	Kaur et al. (2018)
	AK 58	XM13	Higher 5mC and methylation in AK 58 than in XM13	Duan et al. (2020)
Salinity	Kharchia-65	HD2329	Increased 5mC content in CG & CHH in HD2329 in the shoot. Increased 5mC in CG in roots of Kharchia-65	Kumar et al. (2018)
	Dekang-961	Lumai-15	Hypermethylation of CCGG sequence of the tolerant variety	Lan et al. (2009)
	SR3	JN 177	Methylation of stress-responsive gene promoter lower in SR3 than JN177	Wang et al. (2014)
Lead, cadmium & zinc	Pirsabak 2004	Fakhar-e-sarhad	CG DNA hypomethylation at the promoter region of metal detoxification transporter in the sensitive and non-treated controls	Shafiq et al. (2019)
Leaf rust	HD2329 + Lr28	HD2329	The resistant NILs have more hypermethylated and hypomethylated genes than the susceptible NILs	Saripalli et al. (2019)

Lr, Leaf rust resistance

Unconfining DNA methylation analysis in wheat

The enormous size, allohexaploidy with three independently functioning diploid sub-genomes, AA, BB, and DD, which are derived from three diploid progenitors, Triticum urartu, an unknown species related to Aegilops speltoids and Aegilops taushii, respectively, makes the whole process of the study and analysis of the genome to be complex and time-consuming. Although re-sequencing of one accession at a time is more accessible, but the gradual scale-up of the analysis makes it much costlier, which again limits the progress achieved in the epigenomic analysis of the wheat genome. Bisulfite sequencing, being a ubiquitous approach, can help in the efficient analysis of methylation on its own since it discriminates with methylated and unmethylated residues (unmethylated cytosines are converted to uracil after the treatment with bisulfite and later into thymine after performing PCR) (Grehl et al. 2020). However, the sequence capture method accompanied with bisulfite sequencing is more convenient and readable than using it on the whole genome (Whole genome bisulfite sequencing) with less false positives. A part of the sequence can be re-sequenced using probe sets like cDNA, particular chromosome sequences, stress-responsive genes, isoforms, etc., which help in user-defined specific target regions epityping to the reference genome (Afreen and Kumar 2024; Li et al. 2024). Capture assay, which is utilized for DNA sequencing, can help in methyl sequencing, too, which will be cost-effective, and will reduce the sample amount and preparation time. The epitype data thus obtained would widely cover the methylation sites. Moreover, MSAP is used to analyze the global DNA methylation pattern and determine the different digestion patterns, as it works by digesting the DNA with a methylation-sensitive restriction enzyme and further ligating the fragmented DNA to adaptors for amplification (Hermawaty et al. 2024). This, in turn, helps in the determination of positional cloning of the methylated genes (Kumar et al. 2024) where it targets exonic or regulatory regions to find genes impacting the targeted phenotype or trait. Other techniques which could help in a genome-wide methylation profile of wheat is through Methylated DNA Immunoprecipitation Sequencing (MeDIP-seq) (Neary and Carless 2020) which uses antibodies specific to 5-methylcytosine to immunoprecipitate methylated DNA fragments and nanopore sequencing which does not even need the sequence to be bisulfite converted to detect methylated residues (Leduque et al. 2024).

DNA methylation as an avenue for wheat variety improvement

Persistent alterations in the DNA methylation pattern in physiological and stress responses provide several opportunities to improve wheat breeding. Wheat’s differential methylation observed while encountering the biotic and abiotic stresses helps in understanding its local field environmental adaptations (N’diaye et al. 2020). The altered pattern in stresses like chilling, rubbing, cutting, in DMRs and in regulatory genes during biotic stresses increases transcriptional memory, which improves the adaptation of the crop. Thus, it can be deduced that changes in the epigenetic and transcriptional control of duplicated gene copies are among the key contributing elements adding to bread wheat’s expanded formative adaptability and flexibility to diverse environmental constraints.

A prudent and strategic analysis assisted with modern molecular techniques and an integrative study is required, given the complexity of the genome (polyploidy), to establish epigenomics as a promising avenue in producing better wheat crops (Fig. 5). Physiological challenges mandate genome-wide methylation to understand transgenerational and geographical effects on the crop. However, during environmental constraints, gene body/local epigenome profiling of regulatory genes is more beneficial for a conclusive interpretation. Targeted epigenome editing through CRISPR/Cas9, small RNA, gene knockout (Baraskar et al. 2024; Dinkar et al. 2024) or methylation inhibition through chemicals such as 5-azacytidine (Chen et al. 2024) can introduce, delete, or modify specific genes within the wheat’s DNA. Epigenetic priming/stress conditioning (Wang et al. 2016), where wheat can be exposed to sub-lethal doses of stress-indicative or responsive factors, will help the crop to withstand future stress. Similarly, mutant lines with defective methylation machinery (methyltransferase/demethylase mutant) in stress responses will help to identify epigenome-influenced significant genes. These strategies, combined with transcriptomics, proteomics and metabolomics can help to understand the comprehensive impact of methylation to identifying epigenetic markers, which, in turn, will help in marker assisted selective breeding to improve the overall yield. The wheat plant also serves as an ideal model for analyzing polyploidy in plants (Zhang et al. 2022).

Fig. 5.

Strategic approach to improve the wheat breeding program through epigenetics (DNA methylation)

Conclusions

In this review, the use of bibliometric analysis of publications and the retrieved knowledge helped to organize, analyze and report complex data of DNA methylation research in wheat, in a simplified manner. In conclusion, filling the research gap encompassing DNA methylation in wheat is essential for the improvement of its key agronomic traits. Despite the fact that DNA methylation regulates phenotypic and developmental events during stress conditions, the mechanism of action in wheat is not very well investigated. Methylation changes are stable when stress is induced and remain effective even when environmental conditions are back to normal. This could play a boon in inducing methylation in wheat varieties against environmental constraints. The RNA-directed DNA methylation can also behave as a key component of silencing the TEs, which account for 80% of the wheat genome. Remodelling of gene expression of defense-responsive genes could be achieved by using epigenetic editing tools, mainly CRISPR/Cas9 for the heritable and phenotypic changes in wheat. Thus, an elaborated and in-depth study of wheat genome and DNA methylation to incorporate epi mutagenesis in wheat would help to translate DNA methylation into an innovation that is ground-breaking, relevant, and open to wheat breeders for the improvement of new cultivars.

Supplementary Information

Below is the link to the electronic supplementary material.

Data availability

The collected data pertaining to the review have been included in the tables and figures. No additional data are available in the repositories.

Declarations

Conflict of interest

The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.