Functions of RNA N⁶-Methyladenosine Demethylases in Plant Development and Stress Responses

Abstract

N⁶-methyladenosine (m⁶A), the most abundant internal modification in eukaryotic mRNA, regulates gene expression by modulating mRNA metabolism. Demethylases (“erasers”) specifically remove these m⁶A marks. In mammals, FTO and ALKBH5 (ALKBH family members) are key erasers regulating metabolism, reproduction, and development. Notably, heterologous expression of human FTO in rice and potato significantly increase yield. In contrast, research on plant m⁶A demethylases is still in its infancy, though several ALKBH family members have been identified. These enzymes play crucial roles in regulating plant growth and development, as well as in mediating stress responses, highlighting their considerable potential in enhancing crop yield and improving agronomic traits. This review summarizes current knowledge on identified m⁶A demethylases, conducts a phylogenetic analysis of the ALKBH family across representative plant species, and elaborates on the roles of these enzymes in key biological processes such as flowering time regulation, fruit ripening, male fertility, and responses to both biotic and abiotic stresses. Further research on plant RNA m⁶A demethylases will deepen our understanding of RNA epigenetic regulatory mechanisms, uncover valuable genetic resources, and ultimately facilitate the breeding of high-yielding, high-quality crop varieties.

1. Introduction

RNA modification, a post-transcriptional nucleic acid modification, has emerged as a significant area of epigenetic research [1]. Chemical modifications of RNA, including mRNA, rRNA, tRNA, microRNA, and long non-coding RNA, have recently been recognized as powerful mechanisms for gene regulation in various organisms [2]. N⁶-methyladenosine (m⁶A), the most abundant internal modification in eukaryotes, accounts for approximately 80% of all mRNA methylation-related modifications and is the most extensively studied RNA modification [2,3]. m⁶A finely regulates gene expression by influencing mRNA stability, translation efficiency, and degradation, thereby playing a crucial role in diverse physiological activities in plants [4,5,6,7]. As a dynamic and reversible modification, m⁶A is regulated by three enzyme systems: m⁶A methyltransferases (commonly referred to as “writers”), m⁶A demethylases (known as “erasers”), and m⁶A binding proteins (termed “readers”). These enzymes primarily govern RNA fate through the addition, removal, and recognition of m⁶A sites on RNA [8,9,10,11,12]. The identification of these m⁶A-associated enzymes has significantly advanced our understanding of m⁶A’s potential functions and represents a major breakthrough in the field.

Numerous proteins involved in m⁶A modification, including methyltransferases, demethylases, and binding proteins, have been characterized. Among these, demethylases have attracted particular attention. The discovery of m⁶A demethylases has introduced novel perspectives to the field and plays a pivotal role in elucidating the dynamic regulation of m⁶A modification levels within biological systems. To date, research on m⁶A demethylases has predominantly focused on humans and other mammalian models, with FTO (fat mass and obesity associated protein) and ALKBH5 (alkylation repair homolog 5) being the two main demethylases identified [13,14]. They play a central regulatory role in gene expression, cell fate determination, metabolic processes, and disease development through their dynamic regulation of the RNA modification network [13,14,15,16]. Notably, heterologous expression of human FTO in rice (Oryza sativa, a staple food crop), and potato (Solanum tuberosum, an economically important crop) has been shown to significantly increase yield and biomass by approximately 50% [17]. This indicates that regulating m⁶A levels through m⁶A demethylases has significant potential for improving plant growth and increasing crop yields. The high homology of RNA m⁶A demethylases in animals and plants suggests potential similarities in their biological functions, resulting in progressively increasing research on plant m⁶A demethylases. Since the identification of the first plant m⁶A demethylase, such as Arabidopsis ALKBH10B, homologous proteins have been successively identified in various plant species [18,19,20,21,22,23,24]. Studies have demonstrated that these enzymes play essential roles in key processes of plant growth and development, including pollen development, flowering time regulation, fruit ripening, and quality improvement, by precisely regulating m⁶A levels on specific target transcripts [18,25,26,27]. Furthermore, dynamic m⁶A demethylation mediated by these enzymes is established as a core mechanism enabling plants to rapidly activate stress response pathways and enhance tolerance during abiotic (e.g., drought, salinity) and biotic stresses [21,22,23,24,28,29,30,31]. This highlights the great potential of plant m⁶A demethylases as targets for crop improvement. In view of this, systematically reviewing the latest research progress on the regulation of plant m⁶A demethylases in growth, development, and stress response not only helps to deeply understand the molecular mechanism of m⁶A modification dynamic regulation and its core position in plant life activities, but also provides key clues for revealing the epigenetic basis of plant environmental adaptability.

This review focuses on the currently identified plant RNA m⁶A demethylases, presents a comprehensive phylogenetic analysis of the ALKBH family across representative species, highlights their functions in regulating key developmental stages and stress responses, and outlines prospective directions for future research. The primary objective is to provide a theoretical foundation and actionable strategies for utilizing RNA m⁶A demethylases to enhance crop stress resilience and agronomic performance.

2. RNA m⁶A Demethylase

The RNA m⁶A demethylases FTO and ALKBH5 identified in mammals are both nuclear-localized members of the ALKBH family, characterized by their conserved ALKB functional domains. Both enzymes catalyze the demethylation of m⁶A-modified adenosine via a mechanism dependent on α-ketoglutarate and divalent iron ions (Fe²⁺) [11,18]. Compared with animals, m⁶A research in plants remains limited, highlighting the need for further investigation in this field. In recent years, scientists have progressively identified and characterized m⁶A modification-related enzymes in model species such as Arabidopsis thaliana, rice, and tomato (Solanum lycopersicum), as well as in other economically relevant crops, including poplar (Populus), cotton (Gossypium hirsutum), and maize (Zea mays) [18,21,25,28,29,30].

2.1. RNA m⁶A Demethylase in Plants

The RNA m⁶A demethylases in plants exhibit both conservation and diversity, with members of the ALKBH10 subfamily being the most widely distributed. In Arabidopsis, the ALKBH family consists of 14 members (ALKBH1A-D, ALKBH2, ALKBH6, ALKBH7, ALKBH8, ALKBH9A-C, and ALKBH10A-C) [19]. Among these, five members (ALKBH9A/9B/9C/10A/10B) exhibit homology to mammalian ALKBH5 [20], whereas FTO has no known homologs in plants [21]. ALKBH9B was the first plant protein demonstrated to remove m⁶A from single-stranded RNA and Alfalfa mosaic virus (AMV) RNA in vitro [11], marking a significant advancement in plant m⁶A demethylase research. Subsequently, Duan et al. confirmed that ALKBH10B in Arabidopsis can also remove m⁶A from single-stranded RNA in vitro, and the increased m⁶A levels observed in various tissues of the alkbh10b mutants suggest that ALKBH10B functions as an m⁶A demethylase in vivo [18]. AtALKBH6 and AtALKBH8 are considered potential demethylases due to their ability to bind m⁶A labeled RNAs and their involvement in seed germination, seedling growth, and abiotic stress responses [22,23]. Homologs of mammalian ALKBH5 have also been identified in tomato, where they are categorized into ALKBH9 and ALKBH10 subfamilies. SlALKBH2 within the ALKBH9 subfamily, acts as a demethylase affecting fruit ripening [12,21]. OsALKBH9 exhibits RNA m⁶A demethylation activity, removing approximately 50% of m⁶A from synthetic ssRNA or rice mRNA in vitro [25]. Additionally, m⁶A demethylases have been identified in various other plant species, including maize (ZmALKBH10B), poplar (PagALKBH9B and PagALKBH10B), cotton (GhALKBH10 and GhALKBH10B), tea (Camellia sinensis, CsALKBH4A and CsALKBH4B), wolfberry (Lycium barbarum, LbALKBH10), sea buckthorn (Hippophae rhamnoides, HrALKBH10B, HrALKBH10C, and HrALKBH10D), and mustard (Brassica juncea, BjALKBH9B) [28,29,30,31,32,33,34].

Different demethylases exhibit distinct subcellular localizations. ALKBH9B and ALKBH2 are located in the cytoplasm, while ALKBH10B is located in both the cytoplasm and nucleus. This differential localization suggests that these enzymes may target distinct RNA populations, thereby participating in the regulation of diverse aspects of plant biology.

2.2. Phylogenetic Analysis and Functional Annotation of the ALKBH Family in Plants

To date, all identified plant m⁶A demethylases belong to the ALKBH family [11,18]. Using 14 ALKBH protein sequences from Arabidopsis as references, homologous proteins were identified in some representative plant species (Table S1), and a phylogenetic tree of ALKBH gene family protein sequences was constructed using the MEGA 12.0.11. software (Figure 1). Phylogenetic analysis reveals that the members of the ALKBH family are classified into seven distinct subfamilies: ALKBH1, ALKBH2, ALKBH6, ALKBH7, ALKBH8, ALKBH9, and ALKBH10. Each subfamily contains multiple orthologous genes across species, indicating functional conservation throughout plant evolution. Among the ALKBH subfamilies, ALKBH9 and ALKBH10 have been the most extensively studied and exhibit notable gene family expansion, indicating a potential trend toward functional diversification. These subfamilies are considered homologous to animal ALKBH5, which is involved in spermatogenesis and immune responses. In plants, they have been shown to regulate pollen development in rice as well as responses to both biotic and abiotic stresses, indicating that these core biological functions are evolutionarily conserved. In addition, certain members within these subfamilies participate in developmental processes such as flowering time regulation and fruit ripening, further supporting the occurrence of functional divergence. The ALKBH8 subfamily is primarily associated with functions related to product quality improvement, positioning it as a potential target for crop quality regulation. Genes involved in drought and salt stress responses are distributed across the ALKBH6, ALKBH7, ALKBH9, and ALKBH10 subfamilies, reflecting the convergent evolution of abiotic stress response mechanisms. The ALKBH subfamily genes in Arabidopsis (AtALKBH1A-D, AtALKBH9A-C, and AtALKBH10A-B) display clear monophyletic clustering within their respective clades, providing valuable reference points for functional studies in model plant systems. Furthermore, ALKBH members from monocot species, including Zea mays, Oryza sativa, and Brachypodium distachyon, form separate clusters distinct from those of dicots, implying that the ALKBH family may have undergone divergent evolutionary trajectories in monocot and dicot lineages.

2.3. Identification Method of m⁶A Demethylase

2.3.1. Determination of Enzyme Activity In Vitro

To determine whether a candidate protein functions as an RNA m⁶A demethylase, the protein is co-incubated with RNA substrates containing m⁶A in a buffer solution (containing Fe²⁺, α-ketoglutarate, ascorbate, and other cofactors) [18,25]. The change in m⁶A levels within the reaction system was then detected using liquid chromatography–tandem mass spectrometry (LC-MS/MS). A significant reduction in m⁶A levels suggest that the protein possesses RNA m⁶A demethylase activity.

2.3.2. High-Throughput Sequencing Technology

High-throughput sequencing techniques, such as m⁶A sequencing (m⁶A-seq) or Methylated RNA immunoprecipitation sequencing (MeRIP-seq), have been employed to compare m⁶A modification maps between wild-type plants and their corresponding mutants. In this approach, mRNA is fragmented into segments ranging from 100 to 200 nucleotides in length, which are subsequently incubated with m⁶A-specific antibodies. Following immunoprecipitation, the eluted RNA is used for library construction and high-throughput sequencing. Methylated regions are identified as enriched peaks in the immunoprecipitated RNA coverage when compared to the input RNA [35]. Notably, the significant alterations in the methylation levels of numerous m⁶A sites observed in the mutant lines suggest that the target gene may play a role in m⁶A demethylation [18,25]. This method offers a resolution of approximately 200 nucleotides and is relatively straightforward to implement, largely due to the availability of commercial reagents, making it the most widely adopted approach for m⁶A sequencing. In addition, m⁶A selective allyl chemical labeling and sequencing (m⁶A-SAC-seq) is a high-throughput sequencing technique based on enzymatic chemical labeling [36]. First, the m⁶A site is converted to N⁶-allyl adenine by the methyltransferase MjDim 1, catalyzed by the allyl S-adenosylmethionine (SAM) cofactor. Subsequently, N¹–N⁶ cyclization is induced by iodine treatment to form a detectable modified structure. Finally, based on the mismatch signals caused by cyclization sites during reverse transcription, high-throughput sequencing enables single-nucleotide resolution and quantitative detection of m⁶A sites.

2.3.3. Gene Knockout and Overexpress

Following the knockout of a gene encoding a specific ALKBH protein using gene editing technologies such as CRISPR/Cas9, a significantly increased m⁶A modification level in the knockout mutant compared to the wild type may serve as preliminary evidence suggesting that the corresponding protein potentially exhibits demethylation activity. To further validate this hypothesis, a functional complementation experiment can be conducted by introducing the wild-type gene into the mutant. The restoration of both the wild-type phenotype and m⁶A modification levels would provide additional experimental support for the proposed demethylation function of the protein [25]. Furthermore, candidate proteins can be overexpressed to assess whether phenotypes associated with reduced m⁶A levels are induced and to verify whether m⁶A levels are significantly decreased [29].

3. The Role of RNA m⁶A Demethylase in Plant Growth and Development

Heterologous expression of the human FTO gene in rice and potato significantly enhances crop yield by modulating m⁶A modification levels and thereby regulating the expression of associated genes [17]. However, no FTO homologs have been identified in plants to date. All currently characterized plant m⁶A demethylases belong to the ALKBH family, similar to FTO [21]. These enzymes play essential roles in plant growth and development, being involved in key biological processes such as flowering time regulation, fruit ripening, pollen development, and fruit quality improvement.

3.1. Florescence Regulation

The transition from vegetative to reproductive growth in plants is a tightly regulated developmental process. ALKBH10B is the first reported m⁶A demethylase that plays a crucial role in plant growth and development in Arabidopsis [18]. Highly expressed in Arabidopsis flowers, knockout of ALKBH10B leads to delayed flowering—a phenotype attributed to the loss of m⁶A demethylation. Mechanistically, ALKBH10B directly demethylates transcripts of key flowering-time genes, including FLOWERING LOCUS T (FT), SQUAMOSA promoter binding protein-like 3 (SPL3), and SPL9, thereby affecting their mRNA stability and consequently influencing flower development (

Table 1. Plant m⁶A demethylases and their regulatory functions.

Biological Process		Plant Species	m6A Demethylase	Regulate Role	Reference
Development	Florescence	Arabidopsis thaliana	AtALKBH10B	Promote flowering	[18]
	Fertility	Oryza sativa	OsALKBH9	Regulate male reproductive function	[25]
	Fruit Ripening	Solanum lycopersicum	SlALKBH2	Promote fruit ripening	[12]
		Solanum lycopersicum	AcALKBH10	Promote fruit ripening	[27]
		Fragaria vesca	FvALKBH10B	Promote fruit ripening	[37]
	Product Quality	Solanum lycopersicum	AcALKBH10	Improve Product Quality	[27]
		Fragaria vesca	FvALKBH10B	Improve Product Quality	[37]
		Camellia sinensis	CsALKBH4	Improve Product Quality	[31]
Abiotic Stress	Drought	Gossypium hirsutum	GhALKBH10B	Improve drought resistance	[30]
		Zea mays	ZmALKBH10A ZmALKBH10B	Improve drought resistance	[28]
		Hippophae rhamnoides	HrALKBH10B HrALKBH10C HrALKBH10D	Improve drought resistance	[33]
		Poplars	PagALKBH9B PagALKBH10B	Reduce drought resistance	[29]
		Solanum lycopersicum	SlALKBH10B	Reduce drought resistance	[21]
	Salt	Arabidopsis thaliana	AtALKBH10B	Improve salt resistance	[38]
			AtALKBH6	Reduce salt resistance	[22]
			AtALKBH8B	Improve salt resistance	[23]
		Beta vulgaris	BvALKBH10B	Improve salt resistance	[24]
		Poplars	PagALKBH9B PagALKBH10B	Improve salt resistance	[29]
		Solanum lycopersicum	SlALKBH10B	Reduce salt resistance	[21]
Biotic Stress	Virus	Arabidopsis thaliana	AtALKBH9B	Reduce virus resistance	[11]
		Brassica juncea	BjALKBH9B	Improve virus resistance	[34]

). These findings indicate that m⁶A demethylase serves as an important regulator of flowering time in plants [18].

3.2. Affect Fertility

Studies in tomato have shown that decreased m⁶A levels in anthers are associated with abnormal tapetum development and disrupted pollen exine formation, leading to pollen abortion [39]. Similarly, the rice OsALKBH9 mutants exhibit delayed anther tapetum degradation and abnormal pollen exine accumulation, resulting in pollen abortion and complete male sterility. Further investigation revealed that OsALKBH9 directly demethylates TDR and GAMYB transcripts, affecting their mRNA stability and ultimately leading to excessive pollen exine accumulation, thus impacting male fertility in rice [25]. This was the first report implicating that the RNA m⁶A demethylase ALKBH9 is involved in the regulation of male fertility in plants. Conversely, in wolfberry, the expression of XLOC_021201, a homolog of the Arabidopsis m⁶A demethylation gene ALKBH10B, is significantly upregulated in anthers of natural male-sterile mutants compared to fertile lines [32]. Collectively, these findings suggest that different m⁶A demethylases can have diverse effects on plant fertility.

3.3. Fruit Ripening

The mutation of the ALKBH2 gene in tomato results in delayed fruit ripening and a significant increase in m⁶A levels, indicating that SlALKBH2 is essential for the normal ripening of tomato fruits. SlALKBH2 binds to the mRNA of DNA demethylase SlDML2, a key regulator of fruit ripening, and affects the stability of SlDML2 mRNA through m⁶A demethylation, thereby promoting early fruit ripening (Table 1). This finding suggests a close relationship between DNA and RNA modifications [12]. Recent studies have shown that H₂O₂-induced oxidative modifications promote the formation of SlALKBH2 homodimers through intermolecular disulfide bonds, enhancing ALKBH2 protein stability and its function on target transcripts, including SlDML2 [26]. Consistent with this, heterologous expression of kiwifruit (Actinidia chinensis) AcALKBH10 in tomato significantly accelerates the ripening of tomato fruits [27]. Furthermore, in strawberries, FvALKBH10B regulates fruit ripening through the FvABF3-FvALKBH10B-FvSEP3 regulatory cascade [37]. Thus, although RNA m⁶A demethylases from different ALKBH subfamilies exhibit distinct mechanisms in regulating fruit ripening, their function in modulating the ripening process remains highly evolutionarily conserved. This conservation provides potential molecular targets for the precise regulation of fruit ripening timing.

3.4. Improve Product Quality

Transient expression in kiwifruit revealed that the overexpression of AcALKBH10 significantly increased soluble sugar content while decreasing acid accumulation. Conversely, silencing AcALKBH10 resulted in the opposite effects. Similarly, heterologous expression of AcALKBH10 in tomato accelerated fruit ripening and altered fruit flavor by modulating sugar and acid content [27]. Consistent with this, Fvalkbh10b mutant fruit in strawberry displayed markedly reduced concentrations of major sugars (including sucrose, glucose, and fructose) compared to the wild type [37]. Beyond sugar and acid metabolism, studies have shown that CsALKBH4 influences the stability and abundance of transcripts associated with terpene synthesis in tea by removing m⁶A modifications. This action directly affects volatile terpene accumulation and tea aroma [31]. Furthermore, synonymous mutations (synCS) in the coding region of the cucumber CsTRY gene inhibit the function of ALKBH10 by altering mRNA structure, which indirectly leads to abnormal m⁶A modification and downregulation of CsTRY mRNA, and finally promotes the formation of cucumber (Cucumis sativus) domestication traits (smooth fruit) through a recessive epistatic effect [40]. Collectively, these findings suggest that m⁶A demethylases play an important role in mediating m⁶A modification and regulating fruit quality, thereby providing a novel approach for enhancing important characteristics related to fruit quality.

4. RNA m⁶A Demethylase Is Involved in Plant Stress Response

The human FTO homolog has been demonstrated to enhance plant photosynthesis, thereby improving drought tolerance in rice and potato [17]. It is conceivable that m⁶A demethylases not only play a pivotal role in the normal growth and development of plants, but also serve as indispensable factors mediating plant responses to diverse environmental stresses (Table 1).

4.1. Participate in Plant Drought Resistance

RNA m⁶A demethylases exhibit high sensitivity and complexity in the regulation of responses to drought stress. Studies have shown that the cotton demethylase GhALKBH10B increases m⁶A modification and promotes the mRNA degradation of genes related to Ca²⁺ signaling (GhECA1, GhCNGC4, GhANN1, and GhCML13) and Abscisic Acid (ABA) signaling pathways (including GhZEP, GhNCED4, and GhPP2CA). GhALKBH10B functions as a negative regulator of drought resistance in cotton, as evidenced by the enhanced drought tolerance of GhALKBH10B mutants [30]. In maize, demethylase genes ALKBH10A and ALKBH10B, as well as the sea buckthorn demethylase genes HrALKBH10B, HrALKBH10C, and HrALKBH10D, are also significantly upregulated under drought stress, indicating that these plants may reduce m⁶A levels as part of their response to drought stress [28,33]. Transgenic poplars overexpressing PagALKBH9B and PagALKBH10B are more sensitive to drought stress due to decreased proline content [29]. The tomato Slalkbh10b mutant exhibits stronger leaf water retention capacity and higher levels of chlorophyll, soluble sugars, and starch, leading to greater drought tolerance [21]. Transcriptome analysis comparing two near-isogenic maize lines—drought-tolerant ‘Han21’ versus drought-sensitive ‘B73’—revealed that expression levels of m⁶A demethylase genes ZmALKBH10A/ZmALKBH10B are induced by drought stress specifically within the sensitive line ‘B73’, which correlates with an overall decrease in m⁶A modification levels under such conditions [28]. The maize and sea buckthorn homologs of AtALKBH10B positively regulate drought resistance, whereas those from tomato and poplars act as negative regulators (Table 1). These findings indicate that homologs of the same m⁶A demethylase from different species can exhibit divergent responses to drought stress in various plants.

4.2. Participation in Plant Salt Resistance

In Arabidopsis, altered m⁶A deposition under salt stress influences RNA secondary structure, thereby stabilizing mRNAs transcribed from abiotic stress response genes [41]. The expression of AtALKBH9A is significantly induced in Arabidopsis roots under salt stress [42]; however, seeds from AtALKBH6 knockout mutants germinate faster than those of wild-type under similar conditions [22]. The expression of AtALKBH10B is significantly upregulated by ABA, osmotic stress, and salt stress, this effect depends on ABI1, a key component of the ABA signaling pathway. The alkbh10b mutants exhibit significantly lower seed germination rates compared to wild type when subjected to ABA, osmotic, and salt stresses, indicating an increased sensitivity to these stresses during seed germination [38]. Furthermore, in Arabidopsis, overexpression of AtALKBH8B not only enhanced the growth and survival of seedlings under salt stress, but also promoted seed germination and seedling growth in the presence of ABA [23]. BvALKBH6B, BvALKBH8B, and BvALKBH10B are homologs of m⁶A demethylases belonging to the Arabidopsis ALKBH family identified in sugar beet (Beta vulgaris). Under salt stress conditions, the expression of most BvALKB genes changed significant in both leaves and roots, with BvALKBH10B demonstrating a notable upregulation in leaves, suggesting its crucial role in the response to salt stress [24]. Similarly, PagALKBH9B and PagALKBH10B function as m⁶A demethylases in poplar. Transgenic poplars overexpressing these two genes exhibited reduced levels of RNA m⁶A methylation under salt stress. The overexpression led to increased activities of superoxide dismutase (SOD), peroxidase (POD), and catalase (CAT), which subsequently decreased hydrogen peroxide (H₂O₂) accumulation and oxidative damage while protecting chlorophyll, thereby enhancing salt tolerance [29]. The tomato mutant Slalkbh10b exhibited greater resistance to salt attributed to improved leaf water retention capacity, elevated levels of chlorophyll, soluble sugars, and starch, along with upregulated expression of photosynthesis-related genes [21]. Collectively, these findings suggest that RNA m⁶A demethylases play a critical role in plant salt stress adaptation by dynamically regulating the m⁶A modification levels of stress-responsive genes. However, their functions demonstrate high complexity—orthologous genes across species and even paralogs within the same species can mediate diametrically opposite salt tolerance phenotypes. This functional divergence correlates closely with species-specific evolutionary contexts, tissue specificity, and downstream target gene networks. Such functional plasticity provides diverse targets for precision engineering of crop salt tolerance, yet necessitates comparative epitranscriptomics to dissect conserved versus species-specific regulatory pathways.

4.3. Regulate Plant Viral Invasion

Plant viruses pose a serious threat to the growth and development of plants. Previous studies have shown that m⁶A demethylase is involved in regulating plant immune responses to defend against viral infections. m⁶A modification was detected in the genomes of AMV and Cucumber mosaic virus (CMV) [11]. The Arabidopsis AtALKBH9B has demonstrated demethylase activity in vitro, which influences the abundance of m⁶A on the AMV genome. Notably, AtALKBH9B interacts with the capsid protein of AMV within the cytoplasm and modulates viral invasion through its demethylation activity. To confirm this regulatory role, insertion mutants of AtALKBH9B were generated. Following AMV inoculation, wild-type plants exhibited a 100% infection rate, whereas mutants showed only 14%, indicating that AtALKBH9B positively regulates viral invasion. Conversely, in mustard, BjeIF2Bβ recruits BjALKBH9B to confer resistance against radish mosaic virus by altering viral RNA methylation levels [34]. Following tobacco mosaic virus infection, an overall decrease in m⁶A levels was observed in tobacco; concurrently, there was an increase in the mRNA level of XM_009801708—homologous to human ALKBH5—which may be associated with downregulation of m⁶A demethylase activity [43].

Given that the functions of most demethylases remain unclear, and considering their roles vary significantly across plant species and even in response to different stresses within the same species, it is essential to elucidate their contributions to plant growth and stress responses.

5. Discussion and Prospectives

The RNA M⁶A demethylase dynamically regulates mRNA stability, splicing, nucleocytoplasmic transport, and translation efficiency by removing m⁶A methylation modifications, thereby affecting gene expression and playing a key role in various developmental processes [11,18]. The earliest identified animal RNA m⁶A demethylases, FTO and ALKBH5, both belong to the ALKBH family. Notably, heterologous expression of the human FTO gene in rice and potato has been shown to significantly enhance yield and stress tolerance [17]. This important finding has drawn widespread attention and has rapidly spurred intensive research into plant RNA m⁶A demethylases.

Although no homologous gene of FTO has been identified in plants, there are multiple proteins homologous to ALKBH5, such as members of the ALKBH9 subfamily (ALKBH9A/9B/9C) and the ALKBH10 subfamily (ALKBH10A/10B) in Arabidopsis [20]. Among the plant ALKBH family members, ALKBH10B has been most extensively studied. This enzyme is primarily involved in regulating key processes of plant growth and development as well as responses to adverse conditions. However, its functions may vary across different plant species. Accumulating evidence indicates that members of the ALKBH gene family play crucial roles in plant growth, development, and responses to abiotic stresses. Despite the evolutionary conservation among family members, there are substantial differences in their functions and substrate specificities, which may be closely associated with their adaptive evolution in various organisms.

While this study provides insights into the ALKBH family, several limitations remain. Our analysis depends on in silico predictions and data from model plants, lacking experimental validation in most major crops. Additionally, the reliance on low-resolution methods such as MeRIP-seq (~200 nt resolution) limits precise mapping of methylation sites and enzyme-specific targets. Functional redundancy within the ALKBH family further hinders assigning specific biological roles to individual genes. Addressing these challenges will necessitate higher-resolution epitranscriptome profiling, detailed biochemical assays, and analysis of higher-order mutants.

Current research on plant ALKBH demethylases remains narrowly focused, predominantly studying ALKBH9B and ALKBH10B in Arabidopsis [18,38]. In contrast, the identification and functional validation of homologous enzymes in major economic crops—such as rice, maize, wheat, and soybean—remain significantly limited. Future research urgently needs to identify other potential RNA m⁶A demethylases in a wider range of plant species and explore their specific functions in biological processes.

Beyond phenotypic analysis, a critical gap exists in understanding the molecular mechanisms by which m⁶A demethylases recognize and regulate specific target RNAs. Therefore, there is an urgent need to apply single base resolution dynamic detection techniques such as nanopore sequencing, m⁶A RNA immunoprecipitation sequencing (MeRIP-seq) [2], and m⁶A-sensitive RNA-endoribonuclease-facilitated sequencing (m⁶A-REF-seq [44,45], in combination with spatiotemporal expression profiling at the single-cell and tissue levels, to construct high-resolution demethylase-target RNA interaction maps. Concurrently, efforts should focus on identifying interacting proteins (e.g., ubiquitin ligases, kinases) and elucidating how post-translational modifications (e.g., phosphorylation, acetylation) regulate enzyme activity.

Despite the presence of multiple m⁶A demethylase homologs in plants, the mechanisms underlying their functional redundancy and divergence remain poorly understood. Resolving this requires generating multi-gene mutant libraries and employing tissue-specific promoter complementation assays to clarify functional differentiation among family members. Furthermore, an integrated RNA–protein–metabolite multi-omics analytical framework should be established to systematically compare the functional divergence of demethylases across diverse plant groups (e.g., mosses, ferns, monocots, and dicots) and species, thereby uncovering the RNA epigenetic regulatory mechanisms involved in plant adaptive evolution.

The study of plant m⁶A demethylase not only broadens the theoretical framework of RNA epigenetics, but also provides a new perspective for crop breeding. Applying these research findings to crop improvement has the potential to increase crop yield, improve quality, and enhance stress resistance through targeted m⁶A modification, providing innovative strategies and technical support for ensuring food security and sustainable agricultural development.