Recent advances in the plant epitranscriptome
Genome Biology volume 24, Article number: 43 (2023)
Chemical modifications of RNAs, known as the epitranscriptome, are emerging as widespread regulatory mechanisms underlying gene regulation. The field of epitranscriptomics advances recently due to improved transcriptome-wide sequencing strategies for mapping RNA modifications and intensive characterization of writers, erasers, and readers that deposit, remove, and recognize RNA modifications, respectively. Herein, we review recent advances in characterizing plant epitranscriptome and its regulatory mechanisms in post-transcriptional gene regulation and diverse physiological processes, with main emphasis on N6-methyladenosine (m6A) and 5-methylcytosine (m5C). We also discuss the potential and challenges for utilization of epitranscriptome editing in crop improvement.
A variety of naturally occurring chemical modifications on cellular RNAs, collectively termed as epitranscriptome, add an additional layer of regulatory information to RNAs. To date, there are over 170 distinct RNA modifications discovered, with information stored in the MODOMICS database . Over the last decade, advances in techniques in detecting RNA modifications and technologies coupling the next generation sequencing with antibody, chemical, and enzymatic approaches in mapping RNA modification sites have profoundly improved our understanding of the complexity and function of epitranscriptome, especially in messenger RNAs (mRNAs). Thus far, diverse mRNA modifications have been discovered and mapped in eukaryotic cells, including N7-methylguanosine (m7G) and nicotinamide adenine diphosphate (NAD+) modifications  at the 5′-cap, and other modifications occurring internally, such as N6-methyladenosine (m6A) [3, 4], N1-methyladenosine (m1A) [5, 6], 5-methylcytosine (m5C) , N4-acetylcytidine (Ac4C) , 5-hydroxymethylcytosine (hm5C) , m7G , and pseudouridine (ψ) [11, 12]. Accumulating evidence suggests that these modifications with great chemical and structure diversities provide extraordinary regulatory potential in modulating RNA metabolism, thus affecting gene expression. Moreover, extensive characterization of their effector proteins, including writers, erasers, and readers that perform respective functions in installing, removing, and decoding mRNA modifications, has also significantly advanced our knowledge in epitranscriptome regarding its fundamental regulatory roles in diverse and dynamic cellular processes.
In plant epitranscriptome, m6A represents the most prevalent and best characterized internal modification on mRNA. Its landscape and the relevant effectors have been revealed across a variety of plant species [13,14,15,16]. Several other internal modifications, including m5C, m1A, and ψ, have been mapped on a transcriptome-wide scale in some plant species along with identification of their writers (Fig. 1a) [17,18,19]. In addition to these internal modifications, plant mRNAs are also modified at their 5′ end, such as the canonical m7G or non-canonical NAD+ caps (Fig. 1a) [20, 21]. In this review, we highlight recent advances in our understanding of plant epitranscriptome and its regulatory mechanisms in post-transcriptional gene regulation and physiological processes, with main emphasis on m6A and m5C. We also highlight the outstanding questions pertaining to plant epitranscriptome and discuss the potential and challenges of future crop improvement through epitranscriptome editing.
Advances in epitranscriptome profiling technologies
The breakthrough of epitranscriptome studies in the past decade is largely attributed to advancement in epitranscriptome detecting and profiling technologies. Biochemical methods based on physicochemical properties like liquid chromatography-tandem mass spectrometry (LC-MS/MS) [22, 23] allow precise detection and quantification of overall levels of multiple epitranscriptomic marks in plants, including m6A , m1A , m5C, and hm5C . Dot blot analysis using antibodies recognizing specifically modified nucleotides also detects overall RNA modification levels with low sensitivity and precision . However, these approaches are unable to profile the context-dependent transcriptome-wide pattern of RNA modifications. To fill this gap, a variety of high-throughput transcriptome-wide profiling technologies have been developed, including those coupling short-read based sequencing with antibody, chemical, and enzymatic methodologies and Nanopore long-read direct RNA sequencing.
Antibody-, chemical-, and enzyme-based epitranscriptome profiling
Methods coupling next generation sequencing with antibody immunoprecipitation of modified RNAs, including m6A-seq [3, 4], m1A-seq , m5C-seq , hm5C-seq , Ac4C-seq , and m7G-seq  (Fig. 1b), are most commonly used so far for mapping various RNA modifications. These methods have significantly contributed to our current knowledge of the location and distribution of epitranscriptome marks. For instance, m6A-seq has revealed the prevalence of m6A methylation in thousands of transcripts with unique and conserved distribution preferentially around stop codons and in 3′ untranslated regions (UTRs) in eukaryotic organisms [3, 4, 27,28,29]. Nevertheless, these approaches suffer from poor resolution (100–200 nt) on positional information of epitranscriptome marks. Additional steps have been introduced to improve these methods for detecting RNA modifications at single-base resolution. First, incorporating a UV crosslinking step to RNA immunoprecipitation as shown in m6A individual-nucleotide-resolution crosslinking and immunoprecipitation (miCLIP) [30, 31], m6A crosslinking immunoprecipitation (m6A-CLIP) , and photo-crosslinking-assisted m6A sequencing strategy (PA-m6A-seq)  improves m6A detection to the exact or + 1 site. However, these methods are unable to quantify differential modifications among samples without utilizing methylated spike-in controls or an input library for correction or normalization . Second, specific reverse transcriptases are applied to improve the detection resolution for RNA modifications that induce misincorporations during reverse transcription. These methods include m1A-MAP using the reverse transcriptase TGIRT for high-resolution profiling of m1A methylomes . In addition, m6A-seq2 using barcoded adaptors ligated to RNAs from different samples has been developed for simultaneously interrogating m6A dynamics across different samples . This approach could be extended to detect other RNA modifications by using their specific antibodies.
Despite the versatility of antibody-based approaches, their applications have to depend on high-quality antibodies and large quantities of starting RNAs. Alternatively, antibody-independent methods, including bisulfite sequencing (BS-seq) based on chemical-induced signature to detect m5C RNA modifications (Fig. 1b) [7, 37], have been developed for epitranscriptome mark detection. Sodium bisulfite treatment converts an unmethylated cytosine (C) instead of m5C into a uracil (U), creating a signature that allows the mapping of m5C at single-nucleotide resolution. However, a major disadvantage of BS-seq is an unavoidable false positive detection because of incomplete C-U conversion caused by variation of bisulfite treatment, double-stranded RNA structures, or disruption from other modifications such as hm5C [38,39,40,41]. In plants, BS-seq has been applied to detect m5C in tRNA, rRNA and mRNA [42,43,44].
Recent development of enzymatic techniques offers another useful alternative for quantitatively mapping exact locations of m6A in an antibody-independent manner. These techniques either take advantage of m6A-sensitive RNA-cleaving restriction enzymes, such as MAZTER-seq  and m6A-REF-seq , or utilize m6A erasers/readers, such as m6A-SEAL  and DART-seq , or make use of dimethyltransferases like MjDim1 for converting m6A to m62A in m6A-seletive allyl chemical labeling and sequencing (m6A-SAC-seq) . In MAZTER-seq/m6A-REF-seq, the m6A-sensitive enzyme MazF cleaves RNA at ACA rather than m6ACA, thus inferring quantitative site-specific m6A profiles (Fig. 1b). However, as MazF only recognizes the ACA motif in a subset of m6A sites, further engineering of the current enzymes or exploring other m6A-sensitive enzymes is required for expanding the toolbox. Another antibody-free approach DART-seq adopts the fusion protein of the cytidine deaminase APOBEC1 and the m6A-binding YTH domain APOBEC1-YTH, which induces C to U deamination at sites adjacent to m6As  (Fig. 1b). Notably, these antibody-free approaches could work on limited RNAs, as little as nanograms or even picograms, promising m6A profiling in rare materials or single cells. Moreover, many of them are capable of estimating the m6A stoichiometry across different samples. As so far these approaches have been mostly tested in mammalian systems, future optimization and application of these methods in plants will be helpful to explore context-dependent dynamics of plant m6A methylomes.
Nanopore direct RNA sequencing to locate epitranscriptome marks
Although the above-mentioned epitranscriptome profiling approaches have provided unprecedented insights into the distribution and regulation of RNA modifications, challenges remain for quantitative mapping of RNA modification landscape in multiple samples. Moreover, these approaches largely depend on short-read cDNA-based sequencing which requires the conversion of RNA to cDNA. In contrast, the nanopore long-read direct RNA sequencing (DRS) platform is emerging as a promising approach to quantitatively locate and compare RNA modifications at single-nucleotide resolution across different conditions (Fig. 1b) [50, 51]. In this approach, when an RNA molecule traverses a protein nanopore, its modifications cause changes in intensity levels of the electric current, thus permitting prediction of modified bases in a quantitative manner by computational methods [52, 53]. Currently, the algorithms for predicting RNA modifications are built based on either characteristic base-calling error signatures, such as EpiNano  and DiffErr , or machine-learning methods to capture differences in raw current signals, such as Tombo , Nanocompore , and xPore . Nanopore DRS and its associated algorithms have been used to profile RNA modifications, such as m6A, m5C, and ψ, in multiple organisms [56,57,58]. In plants, differential and comparative m6A methylomes at high-resolution have been generated by nanopore DRS for Arabidopsis mutants defective in two m6A writers [31, 59]. Additionally, nanopore DRS and Tombo have been used to identify m5C peaks in Arabidopsis , with an overall pattern similar to that identified by m5C-seq . Future development of algorithms with a focus on improving the accuracy of detecting a broad spectrum of RNA modifications will certainly strengthen epitranscriptome studies.
Besides these experimental approaches, prediction methods, such as RAM-NPPS , BERMP , and PEA , have also been developed to predict m6A in plants. Among these methods, PEA predicts m6A at over 70% sensitivity and specificity in Arabidopsis. Together, all these detection approaches bear their own intrinsic advantages and weaknesses. For instance, different m6A profiling techniques require greatly varied amount of starting materials ranging from 100 ng to 20 μg of mRNA with different detection resolutions and abilities to infer stoichiometric information (Fig. 1c). These characteristics should be considered together with the biological questions to be addressed when designing an epitranscriptome profiling study. In addition, most antibody-free sequencing techniques are yet to be adopted in plant epitranscriptome research despite an increasing trend of using Nanopore DRS to locate the precise RNA modification sites in various plant species. Future applications of these profiling approaches will undoubtedly contribute to our understanding of the dynamics of plant epitranscriptome.
Advances in characterizing epitranscriptome players in plants
m6A writers and recruiters
m6A deposition to target transcripts requires an evolutionarily conserved multicomponent m6A writer/methyltransferase complex. In Arabidopsis, this complex consists of two core methyltransferases, mRNA adenosine methylase (MTA; ortholog of METTL3) and MTB (ortholog of METTL14), and several accessory proteins including FKBP12 INTERACTING PROTEIN 37KD (FIP37; ortholog of WTAP), VIRILIZER (VIR; ortholog of VIRMA), and HAKAI (Fig. 2a and Table 1) [24, 28, 63,64,65,66,67]. Studies in mammals have suggested that the m6A methyltransferase complex could be divided into two subcomplexes, termed m6A-METTL Complex (MAC) and m6A-METTL Associated Complex (MACOM) . MAC formed by MTA and MTB constitutes the catalytic core of the m6A methyltransferase complex, while MACOM containing the accessory subunits including FIP37, VIR, and HAKAI is required for the full activity of MAC. Several additional factors, such as RNA-binding motif protein 15 (RBM15)/RBM15B and zinc finger CCCH domain-containing protein 13 (ZC3H13), are other major components of the mammalian MACOM [69, 70]. In Arabidopsis, a recent study suggests that a HAKAI-interacting zinc finger protein HIZ2 might be the plant equivalent of ZC3H13 , but its biological function in the m6A writer complex needs further investigation. Additionally, although FLOWERING LOCUS PA (FPA) represents the closest ortholog of RBM15/RBM15B and co-purifies with m6A writers, it does not influence global m6A levels when it is defective or overexpressed , implying a limited effect of FPA on m6A modifications in Arabidopsis. Thus, whether plant m6A requires RBM15/RBM15B equivalents needs further exploration.
Within this m6A methyltransferase complex, mutual regulation among different subunits takes place especially at the post-translational level. For instance, WTAP is required for recruiting METLL3-METTL14 to nuclear speckles and mRNA targets in mammalian cells , and for stabilizing the METTL3-METTL14 interaction in Drosophila . ZC3H13 facilitates nuclear localization of other m6A writers in mouse embryonic stem (mES) cells . By contrast, the exact roles of different Arabidopsis m6A writers remain completely obscure. In particular, whether the accessory subunits, FIP37, VIR, and HAKAI, play regulatory functions in mediating m6A methyltransferases, MTA and MTB, awaits further examination. Nevertheless, each individual m6A writer, such as MTA, MTB, FIP37, and VIR, is indispensable for m6A deposition [28, 31, 71, 74], indicating their functional interdependence and mutual regulation for maintaining the functionality of the m6A methyltransferase complex in Arabidopsis.
This Arabidopsis m6A methyltransferase complex, like its mammalian counterpart, installs m6A on mRNAs preferentially near stop codons and in 3′UTRs in a major sequence context of RRACH (R = A/G; H = A/C/U) [13, 28, 31], accounting for the majority of total m6A levels in Arabidopsis. In contrast, another known methyltransferase FIONA1 (FIO1; ortholog to METTL16) acts separately to deposit m6A modifications on a subset of transcripts, contributing modestly to overall m6A levels (Fig. 2a and Table 1) [59, 75]. Unlike METLL16 association with a UACm6AGAGAA sequence embedded in a stem-loop structure [76, 77], FIO1-mediated m6A methylation is enriched in a YHAGA (Y = C/U) motif in coding sequences peaked near stop codons  or motifs resembling RRACH in 3′UTRs . Despite these differences, both METTL16 and FIO1 deposit m6A to the noncoding U6 spliceosomal small nuclear RNA [59, 75, 77].
These two distinct m6A-depositing machineries seem to have both common and distinct targets [59, 75], raising intriguing questions regarding how m6A writers select their targets in response to developmental and environmental signals. Recent advances in plants suggest that target-specificity of m6A could be achieved through recruiting m6A writers to specific transcripts by RNA binding proteins (RBPs) and other writer-associated proteins, which are tentatively named m6A recruiters. The RBP FCA co-purifies with MTA, MTB, and FIP37 and facilitates m6A deposition on the noncoding antisense transcript COOLAIR during Arabidopsis flowering . Another RBP OsFIP37-assocated protein 1 (OsFAP1) recruits the m6A writer OsFIP37 for adding m6A on OsYUCCA3 transcripts during male meiosis in rice (Fig. 2b) . Other characterized m6A recruiters include cryptochrome 2 (CRY2), which undergoes liquid-liquid phase separation (LLPS) to form CRY2-nuclear bodies in response to blue light and interacts with MTA, MTB, and FIP37 to mediate m6A installation on transcripts of central circadian clock oscillator genes (Fig. 2a) . These studies exemplify how various factors are engaged in m6A-depositing machineries to achieve transcript-specific m6A methylation. Specific m6A recruiters may act at different developmental stages or under various environmental stimuli to guide m6A writers to distinct sets of transcripts, generating development- or stimulus-dependent m6A methylomes. Moreover, chromatin or epigenetic signatures also affect m6A deposition in mammalian cells, represented by H3K36me3 that guides m6A methylation co-transcriptionally through METTL14 . Likewise, m6A sites are correlated to H3K36me2 marks in Arabidopsis , implying a potential mechanism of m6A deposition mediated by epigenetic signatures in plants.
Another recent study in rice has suggested the presence of a third m6A methyltransferase ENHANCED DOWNY MILDEW 2-like (OsEDM2L), which contains a highly conserved N6-adenine methyltransferase-like (MTL) domain (Fig. 2b) . Total m6A levels and transcriptome-wide m6A enrichment are significantly reduced in osedm2l mutants, suggesting that OsEDM2L is indispensable for the m6A methylation landscape. As OsEDM2L is specifically expressed in anthers, context-dependent m6A methylation could be modulated by organ-specific m6A methyltransferases.
m6A has been known as a reversible modification since the discovery of m6A erasers Fat mass and obesity-associated protein (FTO) and AlkB homolog 5 (ALKBH5) [93, 94]. Although there are no plant orthologs of FTO, multiple copies of ALKBH5 orthologs have been found in various plant species , including six of them in Arabidopsis, namely ALKBH9A/9B/9C/10A/10B/10C . Among ALKBH5 plant orthologs, ALKBH9B/10B in Arabidopsis and SlALKBH2 in tomato have been characterized to mediate m6A demethylation (Fig. 2 and Table 1) [80, 97, 98]. ALKBH9B not only demethylates m6A-containing viral RNAs and modulates viral infection  but also mediates demethylation of a heat-activated retroelement Onsen in stress granules (SGs) . So far, the mechanisms by which ALKBHs select their targets for demethylation are yet to be elucidated.
Recognition and interpretation of m6A by its readers affect the fate of methylated mRNAs in various mRNA metabolism processes. To date, three groups of m6A readers have been found to recognize m6A-modified transcripts through different mechanisms . YTH domain-containing proteins directly bind m6A through highly conserved YTH domains [100, 101], while heterogeneous nuclear ribonucleoproteins (HNRNPs) recognize m6A-containing RNAs through m6A-dependent RNA structure remodeling [102,103,104,105]. Several other RBPs, such as insulin-like growth factor 2 mRNA binding protein, are associated with m6A-modified transcripts via unknown mechanisms . By now, characterized m6A readers in plants all belong to the YTH domain protein family, including EVOLUTIONARILY CONSERVED C-TERMINAL REGION 2-4 (ECT2-4) and CPSF30L in Arabidopsis and MhYTP2 in apple (Fig. 2 and Table 1) [82,83,84,85, 107, 108].
ECT2/3/4 recognize m6A via their aromatic cages and function redundantly in regulating the timing of organ initiation and leaf morphology [85, 109]. In agreement with their genetic redundancy, ECT3 shares most overlapping target sites with ECT2 and modulates mRNA abundance in the cytoplasm . However, there are conflicting views on functional mechanisms of ECT2. An early study suggests that ECT2 is localized both in the nucleus and cytoplasm to affect 3′UTR length and mRNA stability, respectively , whereas a recent study shows that ECT2 is exclusively localized in the cytoplasm to regulate its target abundance but has little direct effect on alterative polyadenylation (APA) . Another m6A reader CPSF30L forms phase-separated nuclear bodies to influence APA of m6A-containing mRNAs (Fig. 2a) [82, 83, 87]. Disruption of CPSF30L results in global poly(A) site shifts and transcriptional readthrough in recently rearranged gene pairs in Arabidopsis [82, 87]. Additionally, the apple m6A reader MhYTP2 plays dual functions in mediating transcript stability and translation efficiency via unknown mechanisms . Surprisingly, overexpression of MhYTP2 leads to a transcriptome-wide increase in m6A levels possibly via affecting expression levels of multiple m6A writers and erasers, implying possible crosstalk among m6A effectors to maintain appropriate cellular m6A levels.
Notably, plant genomes encode more YTH domain proteins than other eukaryotes. For instance, there are 13 in Arabidopsis, 12 in rice, and 39 in wheat . These genes may exhibit diverse expression patterns under different developmental and stress conditions, thus conferring functional diversities. Further exploration of their biological roles and functional modes are critical for better interpreting m6A epitranscriptome in plants.
Like m6A, m5C deposition, removal, and interpretation in animals require the respective roles of writers including NOL1/NOP2/sun (NSUN) family and DNA methyltransferase homolog DNMT2, erasers such as ten-eleven translocation proteins, and readers including Aly/REF export factor and Y-box binding protein 1 . However, only m5C writers so far have been characterized in plants and include the NSUN orthologs, such as tRNA-specific methyltransferase 4 (TRM4B), TRM4C/NOP2A, TRM4D/NOP2B, and TRM4H in Arabidopsis and OsNSUN2 in rice, and the DNMT2 ortholog tRNA aspartic acid methyltransferase 1 (TRDMT1) in Arabidopsis [19, 42, 43, 111, 112]. Among them, the Arabidopsis TRM4B and rice OsNSUN2 have been shown to mediate m5C methylation in mRNAs (Fig. 2 and Table 1).
Despite sequence homology among plant m5C writer proteins, m5C distribution patterns and targets are less conserved in various plants. For example, as revealed by BS-seq, m5C is evenly distributed in coding sequences (CDSs) and highly enriched in 3′UTRs in Arabidopsis siliques, seedling shoots and roots , whereas m5C is mostly enriched immediately after the start codon in rice seedling shoots . These observations imply functional divergence of m5C writers in selecting their targets in different organisms. Notably, so far different m5C profiling approaches have revealed different m5C distribution patterns. For example, in contrast to those revealed by BS-seq mentioned above , a m5C-RIP-seq study uncovers strong enrichment of m5C in CDSs with a small peak just after the start codon and a high peak before the stop codon in Arabidopsis seedlings , while another m5C-RIP-seq study with a different m5C antibody shows enrichment of m5C in CDSs with a high peak after the start codon and a less pronounced peak before the stop codon . Thus, it is necessary to use other approaches like miCLIP-seq  and Aza-IP-seq  to cross-check bona fide m5C sites in plants. In addition, as functional redundancies among members of NSUN families (e.g. eight in Arabidopsis)  confound the characterization of m5C writers in plants, further elucidation of their biological functions will partly rely on generation and characterization of high-order mutants.
Advances in landscape and regulation of plant m6A dynamics
Characteristics of m6A landscape across plant species
Transcriptome-wide m6A targets and distribution have been extensively profiled with m6A-seq or nanopore DRS in multiple plant species, including pear, apple, strawberry, soybean, pak-choi, Arabidopsis, tomato, rice, maize, etc. [27,28,29, 92, 97, 108, 116,117,118,119,120,121,122,123,124,125,126,127,128,129,130,131,132,133,134]. m6A methylation ratios range from 29% in common wheat to 51% in earthmoss across 13 plant species . The m6A distribution preference around stop codons and in 3′UTRs is highly conserved across most plant species from green algae to higher land plants (Fig. 3a), indicating that m6A is an evolutionarily conserved RNA modification. Despite their low abundance, m6A marks in CDSs have been consistently observed in several plant species, such as pear, strawberry, sea-buckthorn, pak-choi, and rice [92, 116,117,118, 122, 127]. In the rare case, m6A is highest enriched in CDSs in apple leaf . These observations indicate that m6A in CDS might be a previously neglected but important feature with functional significance. For instance, there is an increase in m6A peaks in CDSs during strawberry fruit ripening , implying that m6A in CDS might be associated with changing developmental contexts. In addition, m6A enrichment is also observable around the start codon in Arabidopsis, pear, and sea-buckthorn [28, 116, 118] and within 5’UTRs in apple, pak-choi and sweet sorghum [122, 128, 135].
m6A mainly falls into two sequence motifs in various plant species, including the conserved RRACH motif and the plant-specific URUAY (Y = C/U) motif, whereas a distinct AAACCV (V = A/G/C) motif has only been reported in pak-choi (Fig. 3a) . Interestingly, m6A could occur in divergent sequence contexts at different developmental stages of the same plant species. For instance, in common wheat, the GAGGGAG and UGUAY motifs are found in m6A peaks in grains and leaves, respectively [125, 126].
Dynamic regulation of m6A distribution
m6A methylomes are dynamically changed at different developmental stages and in response to environmental stimuli in diverse plant species (Fig. 3b). Among different Arabidopsis tissues including roots, rosette leaves, and flowers, the fraction of transcripts displaying differential m6A modifications is significantly larger than that showing different transcript levels , indicating that m6A may contribute to organ differentiation.
m6A modifications are also dynamically affected by various stresses. Abiotic stresses, such as salt, drought, and heat, or biotic stresses, such as virus and fungal diseases, do not significantly influence the overall m6A distribution pattern in the 3′UTR and around stop codons but greatly induce dynamic m6A redistribution on selected transcripts [79, 108, 117,118,119, 122, 124,125,126,127,128, 131,132,133, 135, 137,138,139,140,141,142,143,144,145,146,147]. Salt stress significantly increases m6A methylation in Arabidopsis seedlings  and rice shoots but not in rice roots . It induces dynamic deposition of m6A to salt-stress-related transcripts to protect them from degradation in Arabidopsis  and also increases m6A methylation on some salt-resistant-related transcripts to enhance their RNA stability in sweet sorghum . Drought stress causes changes in m6A levels of drought-responsive genes, thereby affecting their expression levels in apple . Cadmium (Cd) induces a transcriptome-wide m6A hypermethylation in barley roots  and alters methylation levels of a large number of transcripts in rice . Together, these observations imply a prominent role of stress-induced m6A redistribution in stress adaptation.
Although many studies have revealed dynamic m6A methylations in organ-, age-, and stress-dependent manners in plants, the underlying mechanisms so far remain elusive. Such dynamic m6A regulation in various contexts could be partially achieved through titration of levels of m6A writers and erasers, resulting in global m6A redistribution. In Arabidopsis, salt stress increases m6A methylation likely through upregulating the expression of the m6A writer genes, MTA, MTB, VIR, and FIP37 (Fig. 3c) . Interestingly, ALKBH10B expression is also upregulated under salt stress , indicating that salt stress-induced m6A dynamics is cooperatively sculpted by increased levels of m6A writers and erasers. Drought stress reduces m6A methylation in sea-buckthorn, which is associated with a significantly increased expression of m6A demethylase genes HrALKBH10B/10C/10D . Besides transcriptional regulation, post-transcriptional modification of m6A effectors or deployment of m6A recruiters under various conditions may also contribute to m6A dynamics. For instance, upon blue light treatment, MTA, MTB, and FIP37 are recruited to the CRY2 nuclear bodies for selective m6A methylation on central oscillator transcripts . Further exploring the mechanisms underlying dynamic m6A alterations on selective transcripts under various conditions will advance our mechanistic understanding of context-dependent regulation of m6A dynamics.
Advances in plant epitranscriptome in gene regulation
RNA modifications determine plant mRNA fate through influencing various aspects of mRNA metabolism, including alternative splicing (AS), APA, folding, translation, localization, transport, and decay (Figs. 2a and 4) [13, 15, 16]. These effects on mRNA metabolism ultimately impact a wide range of physiological processes in plant development and stress responses, as demonstrated by characterization of a collection of the mutants defective in RNA modification effectors.
m6A function in the nucleus
m6A affects the pre-mRNA maturation processes, including AS and APA, to different extents in plants. Although decreased m6A methylation in FIP37-, VIR-, or FIO1-defective Arabidopsis mutants [28, 31, 59, 75] or OsFIP37-defective rice  has a mild effect on transcriptome-wide AS, OsEDM2L-mediated m6A methylation on ETERNAL TAPETUM 1 (OsEAT1) affects AS of OsEAT1, thereby modulating rice anther development (Fig. 4a) . These observations indicate that instead of having a general effect on AS, m6A may regulate AS in specific transcripts or tissues. In contrast, m6A influences APA on a transcriptome-wide scale. Loss of VIR-mediated m6A results in defective 3′end formation of RNAs, which is mainly a shift to usage of proximal poly(A) sites . CPSF30L regulates global poly(A) site choice through recognizing m6A-modified far-upstream sequence but does not show clear preference to proximal or distal poly(A) sites . This m6A-assisted polyadenylation by CPSF30L also restricts transcription readthrough and chimeric mRNA formation at rearranged genomic loci, thus protecting transcriptome integrity . A correlation of m6A sites with APA has also been observed in maize and populus [119, 120], implying a general role of m6A in regulating APA in plants. Moreover, APA at specific transcripts has been linked with plant physiological processes (Fig. 4b). For instance, CPSF30L modulates APA of several transcripts, including SUPPRESSOR OF OVEREXPRESSION OF CONSTANS 1 (SOC1) and NRT1.1 to influence floral transition and nitrate signaling [82, 83]. VIR-mediated m6A methylation affects 3′UTR lengthening via APA of several salt stress regulators including ATAF1, GI, and GSTU17 in stress adaptation .
Besides pre-mRNA maturation, accumulating evidence suggests that m6A exerts important roles in regulating chromatin accessibility and transcription in mammalian cells [150, 151], whereas the similar evidence has just begun to emerge in plants. m6A affects dynamics of FCA nuclear condensates, further influencing the DNA:RNA hybrid (R-loop) formation at the COOLAIR locus in Arabidopsis , which indicates a possible role of m6A in chromatin regulation. Moreover, overexpression of the human FTO in rice increases chromatin accessibility, resulting in a more open chromatin state . Whether m6A mediated by endogenous m6A effectors in plants modulates chromatin states needs further exploration.
m6A function in the cytoplasm
Compared with its nuclear roles in pre-mRNAs, cytoplasmic roles of m6A in affecting mature mRNA metabolism, including folding, translation, localization, and decay, are better understood (Figs. 2a and 4). m6A deposition on transcripts in salt-dependent manner is negatively correlated with RNA folding and the resulting secondary structure, which further affects RNA stability (Fig. 4c) . The m6A-mediated change in RNA secondary structure has also been observed in primary microRNAs (pri-miRNAs), in which MTA-mediated m6A deposition induces their secondary structures . These observations imply that m6A has both stimulatory and inhibitory effects on RNA folding, which may be target- or context-dependent. Structural change in RNAs induced by m6A may affect their interactions with RNA-binding proteins , thus influencing other RNA metabolism steps.
Recent studies have also shown m6A effect on mRNA translation in plants. Combination of m6A-seq and polysome profiling analyses in maize has revealed complex correlations between m6A and translation efficiency, in which m6A is negatively correlated with translation status on the global scale, whereas m6A close to the start codon tends to facilitate translation . Similarly, a m6A site in the 5’UTR of Md4CL3 transcripts in apple has been shown to promote translation , which is similar to the observation in HeLa cells . The promotive effect of m6A on translation has also been observed in apple transcripts containing m6A in the 3′UTR and the transcripts in strawberry fruit [108, 117]. Moreover, m6A-mediated translation seems to have important functions in different physiological processes (Fig. 4d). FveMTA/FveMTB-mediated m6A methylation on the putative ABA receptor (ABAR) facilitates translation of ABAR, thereby regulating strawberry fruit ripening , while the apple m6A reader MhYTP2 enhances translation of MdGDH1L, conferring resistance to powdery mildew . Despite these observations, the mechanisms underlying m6A role in translation remain unknown in plants.
So far, the effect on mRNA stability is the best characterized role of m6A in various plant species and influences multiple aspects of developmental processes and stresses responses. From a transcriptome-wide perspective, m6A displays both promotive and inhibitory effects on mRNA stability in plants. In Arabidopsis, loss of MTA-, VIR-, and FIP37-dependent m6A methylation leads to a global reduction of transcript abundance [28, 31, 140], indicating a stabilizing role of m6A in mRNA. Likewise, m6A readers ECT2/3 stabilize transcripts as most target transcripts of ECT2/3 exhibit reduced abundance upon loss of ECT2/3/4 . These effects on increasing RNA stability likely result from the inhibition of ribonucleolytic cleavage by m6A . Effects of m6A on gene expression have also been examined in various crops via combining m6A-seq and RNA-seq (Fig. 3a). Positive correlation between m6A and transcript abundance has been similarly observed in pear , sea buckthorn , cotton , salt-resistant related transcripts in sweet sorghum , and maize genes bearing 2,4-D-induced m6A peaks , whereas negative correlation has been shown in populus  and green alga . Interestingly, m6A and transcript abundance are generally negatively associated in anthers and during fruit ripening [97, 124] but positively correlated during fruit expansion in tomato . Moreover, m6A near the stop codon and within the 3′UTR is negatively related to transcript abundance, whereas m6A in the CDS tends to stabilize mRNA in strawberry fruit . These complex relationships between m6A and transcript abundance reflect distinct cellular fates of m6A-modified mRNAs in different contexts, implying that mRNA decay mediated by m6A is likely affected by species, developmental stage, cell type, and stress. However, as previous studies have been mostly focused on entire seedlings or organs , the heterogeneity of m6A dynamics and its effect on transcript abundance so far remain elusive. Thus, further analyses, including measuring the half-life of RNA at the transcriptome-wide scale, in various tissues and developmental stages of defective mutants of m6A writers, erasers, or readers will be necessary to disclose the complex and dynamic effects of m6A on transcript stability.
It has also been shown that m6A exerts promotive and inhibitory roles on stability of specific transcripts, which is of great importance in regulating many biological processes in plants (Fig. 4f). In Arabidopsis, FIP37-dependent m6A deposition on WUSCHEL (WUS) and SHOOTMERISTEMLESS (STM) mRNAs accelerates their mRNA decay, which is crucial for maintaining the normal function of shoot apical meristem . The m6A demethylase ALKBH10B demethylates m6A-containing transcripts of FLOWERING LOCUS T (FT) and SQUAMOUSA PROMOTER BINDING PROTEIN-LIKE 3/9 (SPL3/9), thereby increasing their mRNA stability to promote flowering . Likewise, the tomato SlALKBH2 demethylates the m6A-modified SlDML2 transcript and increases its stability, contributing to fruit ripening . Overexpression of an apple m6A reader MhYTP2 promotes the decay of MdMLO19 and MdMLO19-X1, thus promoting apple resistance to powdery mildew . These studies exemplify how m6A destabilizes specific transcripts to regulate various developmental processes and stress responses. By contrast, m6A also stabilizes specific transcripts in multiple physiological processes. In rice, m6A methylation on OsYUCCA3 mediated by the OsFIP37-OsFAP1 complex stabilizes OsYUCCA3 transcripts, thereby promoting local auxin biosynthesis in anthers to secure successful male meiosis and fertility . In strawberry, FveMTA-mediated m6A modifications on NCED5 and AREB1 enhance their transcript stability to ensure normal fruit ripening .
While these pieces of evidence clearly demonstrate that m6A affects transcript stability, the underlying regulatory modes await further examination. Recognition of m6A marks by different m6A readers is possibly important for sorting mRNAs for stabilization or destabilization. It has been suggested that in mammalian cells, YTHDF2 (ortholog of ECT2/3/4) mediates degradation of m6A-containing transcripts via two mechanisms: directly recruiting the CCR4-NOT deadenylase complex for deadenylation of m6A-containing transcripts or interacting with endoribonucleases for endoribonucleolytic cleavage [158,159,160]. On the contrary, expression levels of a majority of ECT2/3-target RNAs are decreased in ect2/3/4 triple mutants, indicating the roles of ECT2/3 in stabilizing transcripts . Intriguingly, upon heat exposure, ECT2 is relocated to SGs through which ECT2 may affect transcript stability . Similarly, the complexes of YTHDFs-m6A-marked mRNAs are partitioned into phase-separated compartments including SGs under stress conditions in mES cells . Many plant ECTs contain low complexity regions or prion-like domains (PrLDs), which could drive LLPS to facilitate formation of membraneless condensates (Table 1) [82, 162], implying that interaction between ECTs and their associated m6A-modified RNAs in distinct cellular compartments might be important for determining the stability of these RNAs.
In addition to the above cytoplasmic roles, m6A may also function in regulating RNA localization. m6A-demethylation of the heat-activated retroelement Onsen mediated by SG-localized ALKBH9B releases Onsen from SG, thus altering its localization (Fig. 2a) . Taken together, current studies have suggested multifaceted nuclear and cytoplasmic roles of m6A in post-transcriptional gene regulation and also raised intriguing questions about the fundamental nature of the relevant mechanisms.
As another abundant internal mRNA modification, m5C has also been studied, to a lesser extent, in recent years, which uncovered its important roles in mediating RNA translation, transport, and stability. m5C is associated with mRNAs with low translational activity in Arabidopsis , whereas m5C mediated by OsNSUN2 facilitates protein synthesis in rice , indicating the functional divergence of m5C in different plant species. In particular, OsNSUN2-mediated m5C on several targets, including OsHO2 and OsPAL1, promotes their protein synthesis, thus enhancing rice adaptation to heat stress (Fig. 4d) . Interestingly, heat stress leads to increased m5C methylation on mRNAs involved in photosynthesis and detoxification systems , implying that m5C is dynamically modulated during stress conditions.
In Arabidopsis, the m5C methyltransferase TRM4B promotes root growth by enhancing mRNA stability of its methylation targets, such as SHORT HYPOCOTYL 2 (SHY2) and INDOLEACETIC ACID-INDUCED PROTEIN 16 (IAA16) (Fig. 4g) [19, 43]. In addition, m5C is highly enriched in mobile mRNAs [58, 113]. TRM4B- and TRDMT1-mediated m5C modifications on mobile mRNAs are essential for systematic mRNA transport from producer to receiver cells over graft junctions (Fig. 4e) .
The last decade has witnessed rapid advances in plant epitranscriptome with respect to m6A dynamics under stresses in different plant species and mechanistic understanding of m6A and m5C modifications in the model plant Arabidopsis and rice. Current evidence strongly suggests that epitranscriptomic marks constitute an essential layer of post-transcriptional gene regulation that determines mRNA fate and ultimately influences plant development and adaptation to various environmental stresses. However, our understanding of plant epitranscriptome is still in its infancy. Many open questions regarding the target selectivity and functional modes of epitranscriptome marks remain to be explored. For example, how do writers and erasers select their targets in different physiological contexts? How are mRNA modifications dynamically regulated in response to environmental stimuli? How do reader proteins recognize their targets and exert their roles in subsequent RNA metabolic processes? Furthermore, since RNA modifications are highly dependent on cellular contexts, the relevant regulatory pathways may differ in different tissues and organs, at distinct developmental stage, or under different stresses. Thus, it is necessary to analyze RNA modification dynamics from tissue levels to cellular levels at single-nucleotide resolution via newly developed profiling techniques. This will advance and expand our knowledge in the plant epitranscriptome.
Multifaced roles of epitranscriptome in developmental processes and stress adaptations in diverse plant species underpin that editing epitranscriptome is a promising strategy for crop improvement. Indeed, introducing the human m6A demethylase in rice and potato not only increases yield but also enhances drought tolerance , demonstrating empirically the great potential of epitranscriptome editing in boosting agricultural production. In particular, advanced DNA/RNA editing techniques in recent years facilitate the development of multiple strategies for epitranscriptome editing of crops [63, 163]. First, modulation of expression or activity of RNA modification-related proteins (RMPs) could be achieved through CRISPR/Cas9-mediated gene editing, thus affecting traits mediated by RNA modifications. Second, RNA modification sites could be directly mutated on specific targets through precise base editors, such as the adenine base editor composed of the catalytically inactive CRISPR/Cas9 protein and an engineered adenosine deaminase causing A to G substitution [164, 165]. Third, RNA modifications could be specifically created or removed on specific target sites through the catalytically inactivated Cas13 (dCas13)-RMPs. For examples, in mammalian cells, dCas13 fused with a truncated methyltransferase domain of METTL3 guides site-specific m6A methylation in target transcripts , while dCas13 fused to m6A demethylases, such as ALKBH5, results in targeted RNA demethylation . Intriguingly, targeted RNA demethylation or methylation can also be achieved in spatiotemporal manners using dCas13 fused with the light-sensitive protein CBIN and its adaptor CRY2 linked to FTO or essential domains of METTL3/14, respectively .
It is noteworthy that the application of epitranscriptome editing in crop biotechnology should at least address the following bottlenecks. First, as m6A sites at single-base resolution are largely unknown in crops, it is necessary to apply techniques, such as miCLIP, MAZTER-seq, m6A-SAC-seq, and Nanopore DRS, to precisely interrogate m6A at single-nucleotide resolution in crops. Second, biological effects of removing/adding epitranscriptome mark at a specific transcript are mostly unknown in plants. Third, although writers, erasers, and readers of RNA modifications have been identified in diverse crops , their endogenous roles and functional mechanisms remain largely elusive. Fourth, DNA/RNA-editing systems and their target specificities and editing efficiencies have yet to be established and examined in most crops. Thus, precise identification of RNA modification sites through advanced profiling approaches, mechanistic understanding of epitranscriptome marks, and development of efficient plant DNA/RNA editors should constitute integral parts of epitranscriptome editing to maximize its potential for crop improvement.
Availability of data and materials
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We apologize to those colleagues whose excellence work could not be cited due to space restrictions.
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Wenjing She was the primary editor of this article and managed its editorial process and peer review in collaboration with the rest of the editorial team.
The review history is available as Additional file 1.
This work was supported by the National Research Foundation Competitive Research Programme (NRF-CRP22-2019-0001), Singapore Food Story R&D Programme (SFS_RND_SUFP_001_04), and the intramural research support from National University of Singapore and Temasek Life Sciences Laboratory.
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Shen, L., Ma, J., Li, P. et al. Recent advances in the plant epitranscriptome. Genome Biol 24, 43 (2023). https://doi.org/10.1186/s13059-023-02872-6