In epigenetics, DNA and histone methylations are critical aspects of genetic regulation. In the past few decades, rapid progress in understanding DNA and histone methylation has been achieved (Greer and Shi, 2012). DNA and histone methylation control different aspects of plants’ development and adaptation. Both these processes are also involved in silencing repetitive elements, which helps maintain genomic stability (Liu et al., 2010; Bartels et al., 2018). In recent years, RNA methylation has emerged as an essential regulatory mechanism in plant epigenetics (Hu et al., 2019a). However, the mechanism of RNA methylation is poorly explored in plants compared to animals. The machinery involved in methylation (writing), demethylation (erasing), and reading are well-documented in animals (Yang et al., 2018). However, the homologs of these writers, erasers, and readers are not thoroughly studied in plants(Hu et al., 2019a). In Arabidopsis, mutations in writers, erasers, and readers of RNA methylation have impacted phenotypic characteristics, showing the importance of RNA methylation in plant growth and development. Transcriptome-wide study on RNA methylation is known as epitranscriptomics. Only a handful of studies are performed on epitranscriptome analysis of plants. Novel RNA methylation marks such as m³C were identified in Arabidopsis using the epitranscriptome approach. The presence of an m³C methylation mark is linked with transcripts’ instability and high turnover rates (Vandivier et al., 2015). Epitranscriptome analysis in callus and leaf tissues of rice reveals the yields of 8,138 and 14,253 m⁶A-modified genes, respectively. Transcription termination and transcription initiation sites exhibited the presence of most of the m⁶A -modified nucleotides (Li et al., 2014). This study in rice reveals the role of m⁶A in gene silencing and activation.

Although RNA methylation plays an essential role in plants, understanding the role of RNA methylation in plants is just beginning. This review has documented a brief overview of current research on plant RNA methylation. Different types of methylation are added to RNA by different methyltransferases and have been studied to various extents. Our review primarily focuses on m⁶A methylation and the machinery involved in its regulation. Here, we discuss current findings, available methods to study RNA methylation in plants, valuable tools to analyze plant epitranscriptome data, limitations, and future prospectus.

RNA methylating enzymes are often considered as writers, whereas readers act as effectors by binding to the methylated nucleotide, and erasers remove the methylation to reset the effect (Lim and Pawson, 2010). In 1994, genes encoding m⁶A writer, methyltransferase-like 3 (METTL3), and METTL14 were recognized in animals (Bokar et al., 1994). Later in 2008 and 2017, the orthologs of METTL3, MTA, and METTL14, MTB, were identified in Arabidopsis (Zhong et al., 2008; Růžička et al., 2017). In Arabidopsis, methyltransferase A/B (MTA and MTB) proteins are involved in embryo development (Hu et al., 2019a). Few other writer proteins like FIP37 (FKBP12 Interacting protein 37), VIR (Virilizer), and HAKAI/CBLL1 (Casitas B-lineage lymphoma-transforming sequence-like protein 1) were also reported in plants (Arribas-Hernández and Brodersen, 2020). In Arabidopsis, FIP37 mutants exhibit massive over-proliferation of stem apical meristem without aerial organs (Shen et al., 2016).

RNA methylation erasers or demethylases are responsible for converting methylated nucleotide to normal. Erasers should be critical to study the role of RNA methylation in plants. In eukaryotes, the erasing of methylation marks are achieved by α-ketoglutarate-dependent dioxygenase (AlkB) homolog (ALKBH) proteins (Zaccara et al., 2019). ALKBH erases alkyl and methyl groups from DNAs, RNAs, and proteins. Interestingly, some ALKBH family members, like ALKBH8, contain both methyltransferase and demethylase activities in animals (Pastore et al., 2012). In Arabidopsis, AtALKBH9B acts as an eraser that removes m⁶A marks from the Arabidopsis and alfalfa mosaic virus (AMV) RNAs during infection to generate host resistance. Inhibition of AtALKBH9B increased the relative abundance of m⁶A marks on AMV RNAs, impairing the systemic invasion of the plant (Martínez-Pérez et al., 2017). Another eraser protein ALKBH10B is noticed to be involved in flowering and vegetative growth maintenance. In Arabidopsis, the alkbh10b mutant delays flowering and vegetative growth repression (Duan et al., 2017).

A mechanism by which methylation affects the expression of RNA is by recruiting methylation loci binding proteins (readers) (Zaccara et al., 2019). In Plants, ECT (EVOLUTIONARILY CONSERVED C-TERMINAL REGION) proteins are methylation loci binding proteins. Among plant nuclear readers, ECT1 and CPSF30 (Cleavage and Polyadenylation Specificity Factor 30) are involved in calcium signaling and abnormal transcription termination, respectively. Among cytoplasmic readers, Arabidopsis ECT2 targets many m⁶A-containing mRNAs, including TTG1 (TRANSPARENT TESTA GLABRA1), ITB1 (IRREGULARTRICHOMEBRANCH1), and DIS2 (DISTORTED TRICHOME2), which are involved in trichome development. It has also been studied that ECT2 is responsible for mRNA stability (Wei et al., 2018). A detailed illustration of plant writers, readers, and erasers is given in Figure 1 . In Arabidopsis, two methyltransferase enzymes, TRM4A and TRM4B are responsible for writing the m⁵C methylation. They are orthologs of human m⁵C methyltransferase. The loss of function mutation in the TRM4A gene does not modify any visible phenotypes. However, the loss of TRM4B reduces root length, implying its role in root growth (David et al., 2017; Hu et al., 2019a). However, erasers and readers of m⁵C are not currently being studied in plants.

Little is known about writers, erasers, and readers in plants; therefore, more future studies are required to elucidate the in-detail role of these proteins in plants.

In plants, m⁶A (6-methyladenosine) methylation was first reported in 1979 in maize, oat, and wheat (Nichols, 1980; Nucleosides and Chromatography, 1980). Till now, m⁶A has been the most studied RNA methylation in plants. Besides m⁶A, m⁵C (5-methylcytosine), pseudouridine, and C-U editing of mitochondrial and chloroplast mRNA are commonly studied in RNA methylation (Arribas-Hernández and Brodersen, 2020).

mRNAs move to distant body parts to potentially act as signaling. A study conducted in Arabidopsis using the meRIP-Seq approach shows that m⁵C modification of mobile RNA modification plays a crucial role in facilitating their transport and presents evidence that mobile m⁵C-modified TCTP1 (TRANSLATIONALLY CONTROLLED TUMOR PROTEIN 1) is translated in target cells and changes root growth (Yang et al., 2019a). In maize, m⁶A methylation shows correlations with the translational status (Luo et al., 2020). In Seagrass, global m⁶A RNA methylation widely contributes to circadian regulation and potentially affects their photo-biological behavior (Ruocco et al., 2020). Additionally, a report on m⁶A methylation in Arabidopsis showed that affects microRNA (miRNA) biogenesis, which demonstrates that m⁶A methylation is necessary to maintain levels of mature miRNAs and their precursors (Bhat et al., 2020). A study on rice has discovered that m⁶A methylation is involved in the pathogenicity of the rice blast fungus Pyricularia oryzae (Shi et al., 2019). Another study in maize discovered that m⁶A methylation is responsible for early-stage callus induction; in this study, genes involved in callus induction, i.e., BABY BOOM and LBD, underwent m⁶A methylation, increasing their expression and promoting callus induction (Du et al., 2020).

Moreover, the fruit of tomatoes showed a molecular link between DNA methylation and RNA methylation (m⁶A) during fruit ripening. In fruits of tomato, the ripening-deficient Colorless non-ripening (Cnr) mutant, which harbors DNA hypermethylation, approximately 1100 transcripts display increased m⁶A levels. Further analysis confirmed that the increase in m⁶A methylation in Cnr mutant fruit is associated with the decreased expression of RNA demethylase gene ALKBH2 (Zhou et al., 2019). A recent study in Arabidopsis shows that m⁶A RNA methylation in flowers and is negatively correlated with gene expression, which limits the activation of heat stress-related genes and compromises fertility during heat (Wang et al., 2022).

Epitranscriptome analysis along with high-throughput annotation of modified ribonucleotides pipeline to identify and classify RNA methylation has predicted different methylation such as 3-methyl cytosine (m³C), 1-methyl guanosine (m¹G), and 1-methyl adenosine (m¹A) in plants. The roles of these marks in photosynthesis, response to cold, osmotic stress, etc., have been predicted using gene ontology analysis (Vandivier et al., 2015; Liang et al., 2020). RNA ribose methylation sequencing (RiboMeth-Seq) is used to detect methylation marks in ribosomal RNAs. A study in Arabidopsis where RiboMeth-seq was used to profile ribosomal RNAs identified 111 cytoplasmic rRNA marks. These identified marks will help study rRNA methylation’s role in plants (Wu et al., 2021a).

The organelle-associated RNAs are highly m⁶A-methylated (98−100% of transcripts in chloroplasts and 86−90% in mitochondria). Around 4-6 m⁶A sites per transcript were identified in both organelles. These methylation marks directly influence gene expression, as the negative correlation between m⁶A methylation and gene expression has been observed in both organelles. The high levels of RNA methylation in chloroplast and mitochondrial RNAs suggest the role of RNA methylation in organelles’ functioning. Considering the importance of chloroplast and mitochondria in photosynthesis and energy metabolism, understanding the role of RNA methylation in these organelles will answer many unsolved questions (Manduzio and Kang, 2021).

A recent study in Arabidopsis thaliana characterized the biological roles of various m⁶A writers (Wong et al., 2023). This study identified the role of m⁶A writers in biological processes such as photosynthesis, stress/defense response, cell growth, metabolic processes, and so on. The research studies explained above are summarized in Table 1 .

The epitranscriptome engineering is a promising tool for achieving food security by editing RNA methylation sites (m⁶A, m⁵C, m¹A, m³C, and so on) through various genome-editing technologies (Shoaib et al., 2022). Furthermore, alternative splicing in plants provides an additional regulatory mechanism and plays key role in plant development (Kadam et al., 2014a; Kadam et al., 2014b; Kadam et al., 2017) nevertheless, impact of RNA methylation on alternatively spliced variants is unknown. In the future, RNA methylation will be a potential target for crop improvement.

An earlier study on maize in 1980 discovered that most of the m⁶A loci are present in the poly-A tail of mRNAs and mainly occur in the R(m⁶A)C (R=A/G) sequence pattern (Nichols, 1980). Based on this study, most of the researchers predicted the role of m⁶A methylation in RNA stability. Later in 2014, a study on Arabidopsis found RR(m⁶A) CH (H=A/C/U) as an extended consensus motif. This study also reveals that m⁶A in Arabidopsis is enriched around the stop codon, within 3′ untranslated regions (3′ UTRs), and around the start codon (Young Hee Choi, 2014). Nanopore direct RNA sequencing of Arabidopsis shows that loss of m⁶A from 3’UTRs is associated with decreased in transcripts accumulation and defective RNA 3′ end formation (Parker et al., 2019). In addition to RRACH motif, UGUAMM and RAGRAG (R=A/G, H=A/C/U, W=A/U, M=A/C) were located as m⁶A motifs in rice (Li et al., 2014). Epitranscriptome analysis suggested the GGAU and URUAY as plant-specific motifs of m⁶A, as they were reported in plants (Anderson et al., 2018; Wei et al., 2018). A methyltransferase enzyme, TRM4, methylates TLS (tRNA-like structure) motifs on specific tRNAs, as a result of methylation, enhances the stability of tRNAs and avoids degradation caused by environmental changes. TLS was also significantly enriched in mobile mRNA datasets of Arabidopsis (Wang et al., 2021a).

However, our knowledge about the occurrence of RNA methylation motifs is incomplete. More single-nucleotide resolution studies are needed to find out motifs enriched for RNA methylation sites.

The methods for studying RNA methylation have been divided into two main parts. 5.1. Sequencing-based methods, where data for sequencing is generated and analyzed. 5.2. Non-sequencing-based methods, where sequencing is not generated.

RNA methylation transcriptome data analysis, i.e., epitranscriptome data analysis, varies depending on the final goal of the experiment. Epitranscriptome data generated using antibody and immunoprecipitation-based techniques follows the same principle as ChIP-Seq (Xu et al., 2020). In ChIP-Seq most commonly used tool is MACS, i.e., model-based analysis of ChIP-Seq, which follows Poisson distribution for peak calling. Similarly, MACS2 is successfully used in epitranscriptome data analysis for methylated RNA peak calling (Gaspar, 2018). MoAIMS toolkit is rapid, efficient, and easy-to-use software implemented in R. MoAIMS (Model-based analysis and inference of MeRIP-Seq) can detect enriched regions of epitranscriptome data efficiently but also evaluate the treatment effect for MeRIP-Seq treatment datasets (Zhang and Hamada, 2020). ExomePeak was one of the earliest methylation peak detection software; along with other toolkits, ExomePeak can also be used for reads mapping, RNA methylation site detection, motif discovery, differential RNA methylation analysis, and functional analysis (Meng et al., 2014). Later, RADAR and MeTDiff were developed, and both these tools have higher sensitivity and specificity than ExomePeak (Cui et al., 2018; Zhang et al., 2019). For those who do not know a computer programming language, m⁶AViewer, a graphical user interface (GUI) platform, is the best option for methylation peak analysis and visualization from sequencing data. m⁶AViewer is a novel m⁶A peak-calling algorithm that identifies high-confidence methylated residues with high precision (Antanaviciute et al., 2017). Another recently developed toolkit, m⁶A Corr, can eliminate the laboratory bias in m⁶A methylation profiles and perform profile to profile comparisons and functional analysis of hyper- (hypo-) methylated genes based on corrected methylation profiles (Li et al., 2020). The deepEA (deep epitranscriptome analysis) is another GUI toolkit with all data analysis functions like data quality check, data filtering, identification of methylation sites, functional annotation, multi-omics integrative data analysis, and prediction analysis based on machine learning. deepEA was developed based on the Galaxy framework. It can be used in windows and Linux (Copy, 2020). The information of all toolkits is summarized in Table 2 . Finally, in the future, more tools will be generated to analyze more sophisticated RNA methylation data.

For most of the RNA methylation experiments, high input of total RNA is required as an abundance of m⁶A is generally less than 0.1% of the total RNA. Most researchers prefer to use mRNA for assay, but recently it has shown that miRNA, tRNA, and snRNA also undergo methylation in plants (Yu et al., 2005; Wu et al., 2021a; Parker et al., 2022). Highly proliferative cells like a callus, shoot apical meristem, and buds show a high rate of RNA methylation than non-dividing cells like leaves, roots, and flowers. So, for non-dividing cells, 100-200 µg of total RNA is required to quantify methylation accurately. For accurate quantification of mRNA methylation, several rounds of mRNA purifications are required for reliable results because mRNA constitutes 1-5% of total RNA, and other RNAs show a high rate of methylation than mRNA. Varios types of RNA methylation reported, but at each antibody-based quantification assay, only one type of RNA methylation can be studied as each antibody detects only one type of RNA methylation. There is no appropriate evidence of whether the same RNA molecules undergo cycles of methylation and demethylation (Arribas-Hernández and Brodersen, 2020).

Several questions in field of plant RNA methylation remain unanswered. For example, how methylation of RNA can regulate gene expression in plants? How methylation affects the stability of cellular RNA in plants? Are reader, writer, and eraser proteins conserved among all plants? Recently, it also became possible to map RNA methylation directly by sequencing native RNAs using nanopore technologies (Begik et al., 2022). This has been applied for the detection of a RNA methylation, such as mapping of genomewide distribution of m⁶A. However, the signal modulations caused by this method is yet to be determined. Addressing these questions will significantly expand our knowledge and broaden the horizons of RNA methylation of plants.

Conceptualization: HS. Writing—original draft preparation: HS, AD, and UK. Writing—review and editing: HS, AD, UK, and JH. Visualization/figures: HS, AD, and UK. Funding acquisition: UK and JH. All authors contributed to the article and approved the submitted version.