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Article

Characterization of the N6-Methyladenosine Gene Family in Peanuts and Its Role in Abiotic Stress

by
Wei Wang
1,2,
Jianxin Bian
1,
Xiaoyu Liu
1,2 and
Xiaoqin Liu
1,2,*
1
Peking University Institute of Advanced Agricultural Sciences, Shandong Laboratory of Advanced Agricultural Sciences in Weifang, Weifang 261325, China
2
College of Life Sciences, Shandong Agricultural University, Taian 271018, China
*
Author to whom correspondence should be addressed.
Int. J. Plant Biol. 2025, 16(1), 7; https://doi.org/10.3390/ijpb16010007
Submission received: 22 November 2024 / Revised: 20 December 2024 / Accepted: 30 December 2024 / Published: 6 January 2025
(This article belongs to the Section Plant Biochemistry and Genetics)
Browse Figures
Figure 1
<p>Role of plant m<sup>6</sup>A gene in stress response.</p> "> Figure 2
<p>Chromosomal distribution of the peanut m<sup>6</sup>A gene. Use the left ruler of the image to estimate the location of the m<sup>6</sup>A gene and the size of each chromosome; genes are shown at the right end of each chromosome.</p> "> Figure 3
<p>Phylogenetic tree of the peanut m<sup>6</sup>A gene. The phylogenetic tree of m<sup>6</sup>A genes were constructed using maximum likelihood (ML). (<b>A</b>) Phylogenetic tree was constructed based on the protein sequences of 22 eraser genes; (<b>B</b>) phylogenetic tree was constructed based on the protein sequences of 19 reader genes.</p> "> Figure 4
<p>Gene structure and conserved domains of the peanut m<sup>6</sup>A gene. (<b>A</b>) The distribution of ten conserved motifs in the m<sup>6</sup>A gene was analyzed by MEME. Different colors represent different patterns and positions; (<b>B</b>) analysis of the gene structure of the m<sup>6</sup>A gene. The yellow rectangle represents the exon, the light green rectangle represents the UTR, and the gray line connecting the two exons represents the intron.</p> "> Figure 5
<p>Collinearity analysis of the peanut m<sup>6</sup>A gene. The red lines represent the presence of collinearity between genes.</p> "> Figure 6
<p>Collinearity of the m<sup>6</sup>A gene among peanut, <span class="html-italic">Arabidopsis thaliana</span> and soybean. (<b>A</b>), Collinearity analysis of the writer gene. (<b>B</b>) Collinearity analysis of the eraser gene. (<b>C</b>) Collinearity analysis of the reader gene. The gray line represents the collinearity between all members, and the red line represents the collinearity between members of the m<sup>6</sup>A gene family.</p> "> Figure 7
<p>Promoter element analysis of the peanut m<sup>6</sup>A gene. Different cis-regulatory elements in the promoter are denoted by square bars of different colors.</p> "> Figure 8
<p>The heatmap presented the expression pattern of m<sup>6</sup>A genes in 15 tissues in peanut. The utilizing row-wise normalization with Z-scores based on previously published RNA-seq data. Main leaf, main stem leaf; Root, roots of 10 days postemergence; Flwr, petals, keel, and hypanthium sepals; AerPeg, elongating aerial pegs; Subpeg, elongating subterranean pegs; ExpPod, Pattee 1 pod; GynStlk, Pattee 1 stalk of gynophore; PodPt3, Pattee 3 pod; PerPt5, Pattee 5 pericarp; SdPt5, Pattee 5 seed; PerPt6, Pattee 6 pericarp; SdPt6, Pattee 6 seed; SdPt7, Pattee 7 seed; SdPt8, Pattee 8 seed; SdPt10, Pattee 10 seed [<a href="#B26-ijpb-16-00007" class="html-bibr">26</a>].</p> "> Figure 9
<p>The qRT-PCR showing the expression levels of 9 m<sup>6</sup>A genes.</p> "> Figure 10
<p>Expression of members of the peanut m<sup>6</sup>A gene under light and mechanical stress. The utilizing row-wise normalization with Z-scores based on previously published RNA-seq data. Peanut pods were immediately wrapped in air-permeable black paper bags to simulate the loss of mechanical stress alone, and D samples collected after 58 h of treatment (day 3) were designated D3. The pods were exposed to air for 58 h (day 3) to simulate darkness and loss of mechanical stress and were designated as sample L3. Two biological replicates were performed for each sample.</p> "> Figure 11
<p>m<sup>6</sup>A protein–protein interaction network.</p> ">
Versions Notes

Abstract

:
Members of the m6A gene family are involved in key biological processes such as plant growth, development, stress responses, and light signal transduction. However, the function of m6A genes in peanuts has been understudied. Our analysis identified 61 m6A family members in the peanut genome, including 21 writer genes, 22 eraser genes, and 18 reader genes, distributed across 20 chromosomes. Phylogenetic analysis revealed that ALKBH proteins are categorized into six subfamilies, while YTH family proteins form nine subfamilies. Promoter cis-element analysis indicated that m6A gene promoters contain light-responsive, hormone-responsive, growth-related, low-temperature defense, and other stress-related elements. Expression studies of AhALKBH8Ba and AhALKBH8Bb in various peanut tissues suggest that these genes play vital roles in peanut fruit needle development. Furthermore, AhETC1a and AhETC1b were significantly upregulated following the loss of mechanical pressure in peanut pods. This study identifies several key genes involved in light and mechanical stress response during peanut pod development.

1. Introduction

Peanuts are an important oil and economic crop, serving as a primary source of edible vegetable oil and protein [1]. The peanut pod, a critical vegetative organ, significantly impacts the yield and quality of peanuts [2]. The unique characteristic of peanut pods is their above-ground flowering and below-ground fruiting, a process requiring a transition from light to dark conditions and changes in mechanical pressure [3]. This developmental process is often accompanied by various biotic and abiotic stresses. Therefore, identifying key regulatory genes involved in peanut pod development is of great importance.
N6-methyladenosine (m6A) is the most prevalent internal chemical modification in eukaryotic mRNAs and plays a vital role in regulating gene expression, including both transcriptional and post-transcriptional processes [4]. m6A modification is a dynamic and reversible process, The m6A methylase (“writer”) adds m6A modifications, the demethylase (“eraser”) removes them, and the methylated reader (“reader”) recognizes them, triggering specific biological functions. m6A includes MTA (homolog of human METTL3), MTB (homolog of human METTL14), FIP37 (homolog of human WTAP) [5], VIRILIZER (VIR) (homolog of human VIRMA), HAKAI, writer complex mounting of and HAKAI-interacting zinc finger protein (HIZ2) (homolog of human ZC3H13) [6], removed by the ALKBH family of erasers [7]. Transcripts carrying the m6A modification can be recognized by a reader, such as a protein containing a YTH domain. By recruiting the reader protein, m6A regulates its biological functions that affect downstream RNA metabolism, including mRNA stability, splicing, translation efficiency, and nuclear export [8,9]. Numerous studies have shown that the m6A gene is essential in biotic and abiotic stresses (Figure 1). In Arabidopsis thaliana, for instance, the transcription and protein levels of ECT8 increase under salt stress, enhancing its binding ability to m6A-modified mRNA, thereby accelerating the degradation of mRNAs that negatively regulate the salt stress response [8]. The m6A-recognition protein ECT8 acts as a molecular sensor for intracellular ABA concentration and directly regulates ABA signaling and the drought stress response [9]. In apples, m6A modification mediated by MdMTA improves the stability of mRNAs involved in oxidative stress, lignin deposition, and drought response, thereby enhancing drought resistance [10]. The m6A writer protein PagFIP37 positively regulates the response of poplar to salt stress by affecting the stability of mRNA under salt stress and the expression of salt-responsive transcripts [11]. In Arabidopsis thaliana, the m6A writer genes MTA, MTB, VIRILIZER (VIR), and HAKAI, mutants of which all exhibit a salt-sensitive phenotype in an m6A-dependent manner [12]. In sorghum, SbMTA and SbALKBH10B-mediated m6A modifications regulate salt tolerance in sorghum by regulating the stability and expression levels of ABA signaling pathways and growth-related genes under salt stress [13]. In peanuts, AhALKBH15 reduces m6A levels, stabilizes the resistance gene AhCQ2G6Y, and enhances resistance to Ralstonia solanacearum [14]. In apple, overexpression of MhYTP2, a homologue of ECT2, enhanced resistance to powdery mildew [15]. These findings highlight the critical role of m6A in various stress responses and plant development, underscoring the importance of investigating the role of the m6A gene in peanut pod development.
Although many studies have explored the function of m6A genes in plants, a comprehensive or systematic analysis of the peanut m6A gene family has not yet been conducted. This gap significantly limits our understanding of peanut m6A genes. Therefore, it is of great significance to study the m6A gene family in peanut and explore its evolutionary and functional differences. To address this, our study presents a whole-genome identification and bioinformatics analysis of the structure and evolution of the m6A gene family in peanuts, providing a systematic and in-depth understanding. We also analyzed the expression patterns of these genes in different tissues and under light and mechanical stress. The aim of this study was to reveal the distribution and gene expression pattern of the m6A gene family in peanut, and to provide clues for further study of their biological functions in peanut pod development.

2. Materials and Methods

2.1. Experimental Materials

The protein sequences of all members of writers and erasers responsible for m6A modification in Arabidopsis were obtained from the TAIR website (https://www.arabidopsis.org; accessed on 20 October 2024). The genome data of Arachis hypogaea cv. Tifrunner, a runner-type peanut (registration number CV-93, PI 644011) [16], was downloaded from https://www.peanutbase.org (20 October 2024).

2.2. Identification of m6A Gene Family Members in Peanut

The protein sequence of the Arabidopsis writer gene was aligned to the peanut genome using blastp to obtain the peanut writer gene’s protein sequence (E-value < 1 × 10−5). The conserved domains of the 2OG_Fell_Oxy (PF03171.23) and YTH (PF04146.18) family were obtained from the Pfam database (https://www.ebi.ac.uk/interpro/entry/pfam; accessed on 25 October 2024). The protein sequences of the 2OG_Fell_Oxy and YTH gene families (E-value < 1 × 10−5) were obtained using HMMER software (Version 3.3.2) to align with the peanut reference genome. All protein sequences were submitted to the CDD website for conserved domain analysis to obtain reliable members of the peanut m6A gene (https://www.ncbi.nlm.nih.gov/Structure/cdd/wrpsb.cgi; accessed on 25 October 2024).

2.3. Molecular Characterization and Chromosomal Localization

Position information of the m6A genes was obtained from the GFF file of the peanut genome. The m6A genes were mapped to the chromosomes using TBtools software (Version 2.142). The amino acid length, molecular weight, and isoelectric point of peanut m6A genes were predicted using the ExPASy website (https://web.expasy.org/protparam/; accessed on 25 October 2024) [17]. Subcellular localization of plant proteins was predicted using http://www.csbio.sjtu.edu.cn/bioinf/plant-multi/#Plant-MPLOC (accessed on 25 October 2024) [18,19,20,21,22].

2.4. Phylogenetic Analysis and Gene Structure Analysis

MEGA software (Version X) was used to construct a phylogenetic tree [using maximum likelihood (ML)] of Arabidopsis thaliana and peanut m6A gene protein sequences [23,24]. Motifs of the m6A gene were analyzed using the MEME website, setting the number of Motif to 10 (https://meme-suite.org/meme/tools/meme; accessed on 27 October 2024). Visualization of gene structures, motifs, and conserved domains using TBtools [25]. The STRING database (https://cn.string-db.org; accessed on 27 October 2024) was used to construct the protein–protein interaction network.

2.5. Predictive Analysis of Promoter Elements

The promoter sequence of 2 kb upstream of the m6A gene member was extracted by TBTools software. These sequences were then submitted to the PlantCARE database for promoter cis-element prediction (https://bioinformatics.psb.ugent.be/webtools/plantcare/html/; accessed on 28 October 2024). Promoter cis-acting elements were visualized using TBtools [25]. Genomic data of soybean (Glycine Max) used for synteny analysis can be downloaded from the following website (https://data.soybase.org/Glycine/max; accessed on 28 October 2024). Interspecies and intraspecies collinearity analyses were performed using TBtools [25].

2.6. RNA-Seq and Bioinformatics Analysis

Clevenger’s previously published transcriptome data were used to analyze the expression of the peanut m6A genes in different tissues. Main leaf, main stem leaf; Root, roots of 10 days postemergence; Flwr, petals, keel, and hypanthium sepals; AerPeg, elongating aerial pegs; Subpeg, elongating subterranean pegs; ExpPod, Pattee 1 pod; GynStlk, Pattee 1 stalk of gynophore; PodPt3, Pattee 3 pod; PerPt5, Pattee 5 pericarp; SdPt5, Pattee 5 seed; PerPt6, Pattee 6 pericarp; SdPt6, Pattee 6 seed; SdPt7, Pattee 7 seed; SdPt8, Pattee 8 seed; SdPt10, Pattee 10 seed [26]. Transcriptome data were used to analyze m6A gene expression in peanut pods under light and mechanical stress [27]. Pattee 3 pods [28] were recovered 10–15 days after insertion into the soil and were named DM. The pods were immediately wrapped in breathable black paper bags and named Sample D (dark). Peanut pods exposed to air were designated as Sample L (light). D (dark and loss of mechanical stress) samples were collected after 10 h, 34 h, and 58 h of treatment and designated as D1, D2, and D3, respectively. L (light and loss of mechanical stress) samples were collected after 10 h, 34 h, and 58 h of treatment and designated as L1, L2, and L3. Pods from samples DM, D1, D2, D3, L1, L2, L3, and seeds from samples D3 (designated as D3S) and L3 (designated as L3S) were analyzed. Two biological replicates were performed for each sample.
For qRTPCR, leaf, stem, root, ExpPod, and PodPt3 were obtained from peanut seedlings grown at Peking University Institute of Modern Agriculture in Weifang, China. Total RNA was extracted using RNAprep Pure Plant Kit (Tiangen, DP432, Beijing, China). CDNA synthesis was performed using FastKing RT Kit (With gDNase) (Tiangen, KR116-02, Beijing, China). The reaction was carried out using ChamQ Universal SYBR qPCR Master Mix (Vazyme, Q711, Nanjing, China) on Applied Biosystems QuantStudio 5 under the following conditions: initial denaturation at 95 °C for 30 s, 40 cycles of denaturation at 95 °C for 10 s, 56 °C for 20 s, and 72 ° C for 20 s, followed by one melting curve cycle between 65–95 °C. Ahtubulin was used as an internal control gene, and three biological replicates of qRT-PCR were performed for each sample. Relative gene expression was determined using the 2−ΔΔCT method.

3. Results

3.1. Physicochemical Properties and Chromosomal Localization of m6A Gene

A total of 61 members of the m6A were identified in the peanut genome, including 21 writer genes, 22 eraser genes, and 18 reader genes. They were named according to their homology with Arabidopsis thaliana genes and their chromosome locations. The m6A phylogenetic component genes are distributed across all 20 chromosomes of peanut. Among them, Chr04, Chr14, Chr06, and Chr16 show higher concentrations, while Chr08 and Chr19 have sparse distributions (Figure 2). The physicochemical properties of the m6A genes were analyzed, showing that the number of amino acids ranges from 180 to 2192, and molecular weight ranges from 19.73 kDa to 240.67 kDa. The theoretical isoelectric points range from 4.81 to 9.55. Subcellular localization predictions of the m6A gene members indicate that AhMETTL4a and AhMETTL4b are located in the cell membrane, while AhFIONA1a and AhFIONA1b are located in the chloroplast. AhFIONA1c is located in both the chloroplast and nucleus, while the other genes are located in the nucleus. Eraser genes are located in the cytoplasm and chloroplast. Reader genes are mostly located in the nucleus, except for AhETC7a, AhETC7b, AhETC7c, AhETC7d, and AhECT1b, which are found in both the cell membrane and nucleus (Table 1).

3.2. Phylogenetic Analysis of the m6A Gene in Peanut

In order to understand the taxonomic status of the m6A gene in peanut. Phylogenetic tree analysis of the m6A gene protein sequences of Arachis hypogaea and Arabidopsis thaliana. Studies have shown that the AtALKBH family proteins of peanut can be divided into six subfamilies (Figure 3A). There are at most 6 homologous genes of AtALKBH1. AtALKBH2 and AtALKBH6 have only two homologous genes in peanut. Similarly, the phylogenetic tree analysis of reader genes showed that the YTH family proteins in peanut could be divided into nine subfamilies. Among them, there are four genes, AhETC7a, AhETC7b, AhETC7c and AhETC7d, which are homologous to AtETC7 in Arabidopsis. It is worth noting that there is no direct homologous gene with AtETC9 and AtETC10 in peanut genome (Figure 3B).

3.3. Analysis of the Gene Structure of Peanut m6A Gene

Analysis of the m6A gene structure showed that members of the same group had similar gene structures. It was found that most of the m6A genes contained more exons and introns except for a few genes such as AhHIZ1, AhHIZ2, AhALKBH8Ba and AhALKBH8Bb (Figure 4B). The protein sequences of m6A members in peanut were analyzed by MEME Suite software (Version 5.5.7). The results showed that the number of motifs varied from 1 to 9. Among the m6A writers, only AhFIP37 genes have more motifs. Other genes contain a small amount of motif. All eraser genes contained motif8, motif2, and motif4 (Figure 4A). In addition to having the same motif, there are some differences in the number and type of motifs between the groups. These differences may lead to different biological functions in peanuts.

3.4. Collinearity Analysis of the m6A Gene Family in Peanut

In this study, most of the m6A genes have a pair of homologous genes on the genome. including AhALKBH10Bc and AhALKBH10Bd, AhALKBH10Bd and AhALKBH10Ba, and AhETC8a and AhETC8b, AhFIP37c and AhFIP37d, AhECT7b and AhECT7c (Figure 5). In order to further study the phylogeny and evolutionary relationship of the m6A gene family in peanut, we analyzed the collinearity of the m6A gene family by constructing a comparative collinearity map of Arabidopsis thaliana, soybean (Glycine Max), and peanut genomes. In the synteny analysis of Arabidopsis thaliana and Arachis hypogaea, writer genes formed 6 orthologous gene pairs; eraser genes formed 14 orthologous gene pairs; and reader genes formed 12 orthologous gene pairs. In the collinearity analysis of Glycine Max and Arachis hypogaea, writer genes formed 32 orthologous gene pairs, erasers gene formed 42 orthologous gene pairs, and reader genes formed 32 orthologous gene pairs. Soybean has higher homology with peanut. Soybean is closer to peanut in phylogeny and evolution (Figure 6).

3.5. Analysis of Cis Elements in the Promoter of Peanut m6A Gene

The cis-regulatory elements of the peanut m6A gene promoter were analyzed. The results showed that the cis elements of the m6A gene promoter mainly included light response elements, hormone response elements, growth and development-related elements and low-temperature defense elements. Except for AhECT7c and AhECT12b, most of the m6A genes contain light-responsive elements (Figure S4). These components mainly include Box4 (ATTAAT), G-Box (CACGTG), and GT1 (GGTTAA). We also found that the reader genes, except AhECT12a, AhECT12b, and AhECT12b, contain a large number of MeJA-responsive elements (Figure S4). SA response elements were found in nearly half of the members. AhFIP3e and AhFIP3e genes contain large number of auxin elements. In addition, a few members have a low-temperature response element, an endosperm expression element, and a defense stress element (Figure 7). These findings suggest that they may play a potential role in plant responses to light signals, stress, and developmental regulation.

3.6. Expression of the m6A Gene in Different Tissues in Peanut

To determine the expression of peanut m6A members in different tissues. Transcriptome analysis was performed on 15 tissues of peanut. The results showed that there were similar expression patterns among homologous genes. The expression level of the m6A gene was lower at the flower and GynStlk stages. More than half of the m6A-regulated genes among the writer and reader regulated genes were consistently highly expressed in different tissues of peanut seedlings, with only a few moderately or lowly expressed (Figure 8). This indicates that peanut needs to maintain its m6A modification level during growth and development. Writer members AhFIONA1a and AhMETTL4b were highly expressed at the ExpPod stage of pod expansion (Figure 9). At the same time, we found that eraser genes showed obvious tissue specificity. It is worth noting that AhALKBH8Ba and AhALKBH8Bb are almost not expressed in leaves, flowers, roots and peanut pods. However, the expression levels of AerPeg, Subpeg, ExpPod, GynStlk and PodPt3, PerPt5, PerPt6 were higher in the process of peanut needle penetration. Reader member AhECT1a was highly expressed in ExpPod and PodPt3 (Figure 8 and Figure 9). Correlation analysis showed that there was a positive correlation between the expression of writer genes. AhALKBH10Bb, AhALKBH10Bd, AhALKBH8Ba, AhALKBH8Ba were negatively correlated with the expression of other eraser genes. The expression of AhECT8a and AhECT8b in reader genes was negatively correlated with AhECT1a, AhECT1b, AhECT4a and AhECT4b. There was a positive correlation between gene expression in different tissue sites (Figure S5). The different expression patterns of these genes reflect the complexity of regulatory genes in peanut growth. The potential biological function of the m6A gene was explored by analyzing its specific expression in different tissues of peanut.

3.7. Expression of the m6A Gene Under Light and Mechanical Pressure

In order to explore the expression of the m6A gene under light and mechanical pressure. The transcriptome of peanut pods treated with light and mechanical pressure was analyzed. Among eraser genes, only AhALKBH2a, AhALKBH9Aa, AhALKBH9Ba and AhALKBH9Bb were significantly upregulated under light induction. There was no significant change in the expression of other genes. There was no obvious change in the expression of reader genes induced by light. For writer genes, AhMETTL4a, AhMTAa and AhMTAb genes were significantly upregulated under light induction. Meanwhile, the expression of AhFIP37f gene was slightly downregulated under light induction. In the absence of mechanical pressure, the expression of most of the writer genes did not change. Only the expression of AhMETTL4a gene was significantly upregulated. YTH family genes are essential m6A marker decoders, and most of the reader genes are significantly upregulated after loss of mechanical pressure. In particular, AhETC1a and AhETC1b genes were significantly upregulated (Figure 10).

3.8. Interaction Network of Peanut m6A Protein

In order to explore the protein–protein interactions between m6A proteins and other proteins, we used STRING database to construct the protein–protein interaction network. The network consists of 26 nodes and 84 protein interactions, with average node degree of 6.46 and a value of 0.478 for avg. local clustering coefficient. Most of the genes in the protein–protein interaction network are m6A genes. These include 4 writer genes, 7 eraser genes and 15 reader genes. AhMTAa, AhMTAb, AhMTBa, AhMTBb, AhECT12a and AhECT12b genes have more interactive proteins. In addition, most of the reader genes can interact with AhMTAa, AhMTAb, AhMTBa, and AhMTBb proteins. AhALKBH1Aa, AhALKBH1Ab, AhALKBH1Ca, AhALKBH1Cb, AhALKBH1Da, and AhALKBH10Bb proteins can interact with AhMTAa, AhMTAb proteins (Figure 11).

4. Discussion

m6A genes are crucial components of the epigenetic network regulating plant growth, development, and responses to abiotic stress, playing key roles in post-transcriptional modification [4]. While m6A genes have been extensively studied in Arabidopsis thaliana, tomato [29], cotton [30], and sorghum [31], their role in peanuts remains underexplored. In this study, we identified 61 m6A genes in the peanut genome through bioinformatics, including 21 writer, 22 eraser, and 19 reader genes. Subcellular localization showed that m6A genes were distributed across the cell membrane, cell wall, chloroplast, cytoplasm, Golgi apparatus, and nucleus, suggesting diverse biological functions. Peanut pod development, which requires hormonal and dark environments [32,33], may also involve m6A regulation, as indicated by the presence of hormone- and light-responsive elements in the promoters of m6A genes. Most reader genes, except AhECT12a, AhECT12b, and AhECT12c, contained MeJA-responsive elements, and nearly half had SA response elements, suggesting a role in pathogen resistance. AtCPSF30 is a homolog of human HyTDC, a cleavage and polyadenylation specific factor that functions in nitrate signaling [34]. In Arabidopsis thaliana, YTH03, YTH05, and YTH10, which are homologous to AtETC1, AtETC2, AtETC3, and AtETC4 specifically bind to m6A-containing RNA in rice. Knockout of YTH03, YTH05, or YTH10 caused a decrease in plant height. Further studies showed that YTH03, YTH05, and YTH10 were simultaneously knocked out, resulting in a severe dwarf phenotype. The three genes are functionally redundant in regulating the plant height of rice [35]. The genes with similar conserved domains in peanut are close in evolutionary relationship, and there may be redundancy. The evolutionary relationship between different domain genes is distant. These differences may lead to different biological functions in peanuts. Protein interaction network found that AhALKBH1Aa, AhALKBH1Ab, AhALKBH1Ca, AhALKBH1Cb, AhALKBH1Da proteins can interact with AhALKBH12a proteins. These genes have the same expression pattern in different tissues. At the stage of pod enlargement, the expression levels of ExpPod, PodPt3, PerPt5 and PerPt6 were upregulated. This suggests that these genes are involved in the biological process of peanut pod expansion through interaction.
Studies show that m6A genes are involved in plant development and stress responses [10,35,36,37,38]. Under ABA treatment, the ALKBH9B gene removes m6A modifications from ABI1 and BES1 transcripts, increasing their stability and promoting seed germination and seedling development [37]. In kiwifruit, overexpression of AcALKBH10 significantly increases soluble sugar content and decreases acid accumulation, while heterologous expression of AcALKBH10 in tomato accelerates fruit ripening [39]. In cotton, the alkbh10B mutant increases m6A levels, improving the stability of genes related to ABA synthesis (GhZEP, GhNCED4, GhPP2CA) and Ca2+ signaling (GhECA1, GhCNGC4, GhANN1, GhCML13), thereby enhancing drought tolerance [40]. In poplar, overexpression of PagALKBH9B and PagALKBH10B increased SOD, POD, and CAT activities, improving salt tolerance [7]. However, there are few reports on the role of the m6A gene in light stress. Among eraser genes, only AhALKBH2a, AhALKBH9Aa, AhALKBH9Ba and AhALKBH9Bb were significantly upregulated under light induction. There was no obvious change in the expression of reader genes induced by light. In foxtail millet, SiYTH1 knockout mutants are sensitive to drought, showing reduced stomatal closure and increased H2O2 accumulation, while overexpression of SiYTH1 enhances drought resistance [41]. In wheat, YTH gene expression was significantly altered under abiotic stress [42]. In Arabidopsis thaliana, ECT2 affects trichome development by binding m6A-modified transcripts of trichome-related genes ITB1, TTG1, and DIS2 [43]. AhECT1a, AhECT1b, AhECT4a and AhECT4b were significantly upregulated during peanut pod enlargement. AhECT1a, AhECT1b, AhECT4a YTH family genes are essential m6A marker decoders, and most of the reader genes are significantly upregulated after loss of mechanical pressure. In particular, AhETC1a and AhETC1b genes were significantly upregulated. We identified several candidate m6A genes potentially involved in peanut pod development, providing a foundation for further functional studies. At the same time, the interaction of peanut m6A protein was studied to better understand the molecular mechanism of the m6a gene in the process of performing biological functions.

5. Conclusions

In this study, we first performed a comprehensive and systematic analysis of the m6A gene family in peanut. A total of 61 genes were identified and renamed to better understand the underlying gene functions—21 write genes, 22 erase genes and 18 reader genes, distributed on 20 chromosomes. Phylogenetic analysis showed that ALKBH proteins were divided into six subfamilies and YTH proteins into nine subfamilies. Comparing phylogenetic trees and dividing them into subclades can help to distinguish the functions of different subfamilies. Promoter cis-element analysis showed that the promoter of the m6A gene contained light-responsive elements, hormone-responsive elements, growth-related elements, low-temperature defense elements and other stress-related elements. Gene expression patterns showed that most of the genes had a wide range of tissue expression, and the expression of AhALKBH8Ba and AhALKBH8Bb in different tissues of peanut indicated that these genes played an important role in the development of peanut fruit needles. Furthermore, AhETC1A and AhETC1B were significantly upregulated after loss of mechanical pressure in peanut pods. Our study lays the foundation for a better understanding of the functional characteristics of the peanut N6-methyladenosine gene family.

Supplementary Materials

The following supporting information can be downloaded at: https://www.mdpi.com/article/10.3390/ijpb16010007/s1. Figure S1: The top ten motif sequence logos of Writer gene members; Figure S2: The top ten motif sequence logos of Eraser gene members; Figure S3: The top ten motif sequence logos of Reader gene members; Figure S4: Statistics of the number of elements on the promoter of peanut m6A gene; Figure S5: Correlation analysis of m6A gene expression in peanut.

Author Contributions

Conceptualization, X.L. (Xiaoqin Liu) and W.W.; methodology, W.W., J.B. and X.L. (Xiaoyu Liu); software, W.W., J.B. and X.L. (Xiaoyu Liu); formal analysis, W.W., J.B. and X.L. (Xiaoyu Liu); data curation, W.W.; writing—original draft preparation, W.W. and X.L. (Xiaoqin Liu); writing—review and editing, X.L. (Xiaoqin Liu); funding acquisition. All authors have read and agreed to the published version of the manuscript.

Funding

This research was supported by the Taishan Scholars Program (tsqn202103161), Key R&D Program of Shandong Province, China (2024LZGC035) to X.L. (Xiaoqin Liu).

Data Availability Statement

No data were used for the research described in this article. Data sharing is not applicable to this article.

Conflicts of Interest

The authors declare no conflicts of interest.

References

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Figure 1. Role of plant m6A gene in stress response.
Figure 1. Role of plant m6A gene in stress response.
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Figure 2. Chromosomal distribution of the peanut m6A gene. Use the left ruler of the image to estimate the location of the m6A gene and the size of each chromosome; genes are shown at the right end of each chromosome.
Figure 2. Chromosomal distribution of the peanut m6A gene. Use the left ruler of the image to estimate the location of the m6A gene and the size of each chromosome; genes are shown at the right end of each chromosome.
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Figure 3. Phylogenetic tree of the peanut m6A gene. The phylogenetic tree of m6A genes were constructed using maximum likelihood (ML). (A) Phylogenetic tree was constructed based on the protein sequences of 22 eraser genes; (B) phylogenetic tree was constructed based on the protein sequences of 19 reader genes.
Figure 3. Phylogenetic tree of the peanut m6A gene. The phylogenetic tree of m6A genes were constructed using maximum likelihood (ML). (A) Phylogenetic tree was constructed based on the protein sequences of 22 eraser genes; (B) phylogenetic tree was constructed based on the protein sequences of 19 reader genes.
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Figure 4. Gene structure and conserved domains of the peanut m6A gene. (A) The distribution of ten conserved motifs in the m6A gene was analyzed by MEME. Different colors represent different patterns and positions; (B) analysis of the gene structure of the m6A gene. The yellow rectangle represents the exon, the light green rectangle represents the UTR, and the gray line connecting the two exons represents the intron.
Figure 4. Gene structure and conserved domains of the peanut m6A gene. (A) The distribution of ten conserved motifs in the m6A gene was analyzed by MEME. Different colors represent different patterns and positions; (B) analysis of the gene structure of the m6A gene. The yellow rectangle represents the exon, the light green rectangle represents the UTR, and the gray line connecting the two exons represents the intron.
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Figure 5. Collinearity analysis of the peanut m6A gene. The red lines represent the presence of collinearity between genes.
Figure 5. Collinearity analysis of the peanut m6A gene. The red lines represent the presence of collinearity between genes.
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Figure 6. Collinearity of the m6A gene among peanut, Arabidopsis thaliana and soybean. (A), Collinearity analysis of the writer gene. (B) Collinearity analysis of the eraser gene. (C) Collinearity analysis of the reader gene. The gray line represents the collinearity between all members, and the red line represents the collinearity between members of the m6A gene family.
Figure 6. Collinearity of the m6A gene among peanut, Arabidopsis thaliana and soybean. (A), Collinearity analysis of the writer gene. (B) Collinearity analysis of the eraser gene. (C) Collinearity analysis of the reader gene. The gray line represents the collinearity between all members, and the red line represents the collinearity between members of the m6A gene family.
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Figure 7. Promoter element analysis of the peanut m6A gene. Different cis-regulatory elements in the promoter are denoted by square bars of different colors.
Figure 7. Promoter element analysis of the peanut m6A gene. Different cis-regulatory elements in the promoter are denoted by square bars of different colors.
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Figure 8. The heatmap presented the expression pattern of m6A genes in 15 tissues in peanut. The utilizing row-wise normalization with Z-scores based on previously published RNA-seq data. Main leaf, main stem leaf; Root, roots of 10 days postemergence; Flwr, petals, keel, and hypanthium sepals; AerPeg, elongating aerial pegs; Subpeg, elongating subterranean pegs; ExpPod, Pattee 1 pod; GynStlk, Pattee 1 stalk of gynophore; PodPt3, Pattee 3 pod; PerPt5, Pattee 5 pericarp; SdPt5, Pattee 5 seed; PerPt6, Pattee 6 pericarp; SdPt6, Pattee 6 seed; SdPt7, Pattee 7 seed; SdPt8, Pattee 8 seed; SdPt10, Pattee 10 seed [26].
Figure 8. The heatmap presented the expression pattern of m6A genes in 15 tissues in peanut. The utilizing row-wise normalization with Z-scores based on previously published RNA-seq data. Main leaf, main stem leaf; Root, roots of 10 days postemergence; Flwr, petals, keel, and hypanthium sepals; AerPeg, elongating aerial pegs; Subpeg, elongating subterranean pegs; ExpPod, Pattee 1 pod; GynStlk, Pattee 1 stalk of gynophore; PodPt3, Pattee 3 pod; PerPt5, Pattee 5 pericarp; SdPt5, Pattee 5 seed; PerPt6, Pattee 6 pericarp; SdPt6, Pattee 6 seed; SdPt7, Pattee 7 seed; SdPt8, Pattee 8 seed; SdPt10, Pattee 10 seed [26].
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Figure 9. The qRT-PCR showing the expression levels of 9 m6A genes.
Figure 9. The qRT-PCR showing the expression levels of 9 m6A genes.
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Figure 10. Expression of members of the peanut m6A gene under light and mechanical stress. The utilizing row-wise normalization with Z-scores based on previously published RNA-seq data. Peanut pods were immediately wrapped in air-permeable black paper bags to simulate the loss of mechanical stress alone, and D samples collected after 58 h of treatment (day 3) were designated D3. The pods were exposed to air for 58 h (day 3) to simulate darkness and loss of mechanical stress and were designated as sample L3. Two biological replicates were performed for each sample.
Figure 10. Expression of members of the peanut m6A gene under light and mechanical stress. The utilizing row-wise normalization with Z-scores based on previously published RNA-seq data. Peanut pods were immediately wrapped in air-permeable black paper bags to simulate the loss of mechanical stress alone, and D samples collected after 58 h of treatment (day 3) were designated D3. The pods were exposed to air for 58 h (day 3) to simulate darkness and loss of mechanical stress and were designated as sample L3. Two biological replicates were performed for each sample.
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Figure 11. m6A protein–protein interaction network.
Figure 11. m6A protein–protein interaction network.
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Table 1. Analysis of physicochemical properties of members of the m6A gene in peanut.
Table 1. Analysis of physicochemical properties of members of the m6A gene in peanut.
m6A MemberGene ID (arahy.Tifrunner.
gnm2.ann1.)		Chr.StartEndStrandNumber of Amino AcidsMolecular Weight/DaPISubcellular
Location
AhFIONA1aMR7L4V.1Chr03140,142,325140,145,261+49454,987.677.62Chloroplast.
AhFIONA1cUN7M5W.1Chr13142,229,486142,236,603+70779,057.308.91Chloroplast.
AhFIONA1bDZ4B2B.1Chr1113,681,61513,686,002+25227,829.109.18Chloroplast.
Nucleus.
AhMTAbYTN4Z3.1Chr122,671,5212,676,437+75483,846.576.08Nucleus.
AhMTAa3PG13R.1Chr022,448,3262,453,309+72180,119.135.97Nucleus.
AhMETTL4bK9WZZ6.1Chr16150,599,745150,604,141+42548,927.608.32Cell membrane.
AhMETTL4a4B2PQ4.1Chr06118,332,786118,337,182+42548,927.608.32Cell membrane.
AhMTBbJBBQ4L.1Chr19159,149,773159,155,400−1144126,171.027.90Nucleus.
AhMTBaIW4QJC.1Chr09108,973,001108,976,901+57562,961.287.21Nucleus.
AhFIP37bNDZJ5A.1Chr0345,795,00545,801,158−33938,426.215.01Nucleus.
AhFIP37hT862AX.1Chr18116,828,851116,835,273+34138,435.455.60Nucleus.
AhFIP37gGAE861.1Chr1349,069,39249,075,239−36741,742.215.24Nucleus.
AhFIP37eP2M8BS.1Chr0841,769,45441,774,119−35139,706.925.60Nucleus.
AhFIP37aBAEJ4E.1Chr0191,586,58191,594,025+40845,316.654.81Nucleus.
AhFIP37dV3MNVK.2Chr07152,527159,830+34338,973.868.08Nucleus.
AhFIP37f9FBM9V.1Chr11101,115,539101,121,935−34239,265.546.25Nucleus.
AhFIP37cN3JE1D.2Chr0343,475,94843,485,452−47254,023.928.29Nucleus.
AhVIRILIZERaC5PQVK.1Chr02100,930,256100,943,875+2192240,668.035.37Nucleus.
AhVIRILIZERbS879XT.1Chr12118,183,955118,197,565+2175238,626.515.36Nucleus.
AhHIZ1aU0SE7F.1Chr051,096,7041,099,157−37639,536.137.12Nucleus.
AhHIZ1bBDLH6M.1Chr151,096,7041,099,157−37639,536.137.12Nucleus.
AhALKBH1AbDXN1WG.1Chr2012,622,08712,625,338+35740,591.226.53Cytoplasm.
AhALKBH1AaP16M7J.1Chr107,114,4787,117,721+35740,465.116.53Cytoplasm.
AhALKBH1Ca8PX6PB.1Chr061,508,8421,510,917−32135,350.029.10Nucleus.
AhALKBH1Cb4SYW8G.1Chr1625,729,58525,730,741+33637,113.229.07Nucleus.
AhALKBH1DbR859Y3.1Chr12107,793,516107,796,063−31135,182.669.45Chloroplast.
Cytoplasm.
AhALKBH1DaX2XVT6.1Chr0293,082,44893,084,961−31135,092.639.55Chloroplast.
AhALKBH2aTFI8LM.1Chr047,135,0077,137,625−23727,164.849.23Chloroplast.
AhALKBH2bHFV92P.1Chr148,575,4618,578,100−23727,204.959.23Chloroplast.
AhALKBH6aF1EX5J.3Chr0588,338,77688,348,515−55762,843.116.82Nucleus.
AhALKBH6b75P5EB.1Chr15155,411,315155,425,900+55462,883.856.35Nucleus.
AhALKBH8AaE4PV4B.1Chr052,700,0402,701,844−34638,499.916.53Chloroplast.
AhALKBH8AbC0VXQN.1Chr152,700,0402,701,844−34638,499.916.53Chloroplast.
AhALKBH8Ba68R93N.1Chr073,332,1053,332,764+21925,091.485.47Nucleus.
AhALKBH8BbIELT58.1Chr173,951,7843,952,461+22525,758.075.13Nucleus.
AhALKBH9BaZUG4EU.1Chr06113,352,347113,356,457−50056,400.106.95Nucleus.
AhALKBH9Bb2M720R.1Chr16146,329,818146,334,126−56563,620.865.52Nucleus.
AhALKBH9AaU5HZVG.1Chr106,308,1876,311,328−39144,578.808.85Cytoplasm.
AhALKBH9AbG66ML0.1Chr2011,539,27711,542,737−42048,037.809.14Cytoplasm.
AhALKBH10BaUA6S11.1Chr0298,218,38698,227,226+53258,222.505.79Cytoplasm.
AhALKBH10BcSPY99B.1Chr12114,482,265114,491,151+53258,164.425.78Cytoplasm.
AhALKBH10Bb45BME0.1Chr0613,886,18213,895,730−58263,490.536.04Cytoplasm.
AhALKBH10BdIEB1S3.2Chr166,142,1226,149,906+51856,568.655.74Chloroplast.
Cytoplasm.
AhECT7b18BCEG.1Chr0411,627,26711,632,571+66172,758.107.04Cell membrane. Cell wall.
Chloroplast.
Cytoplasm. Golgi apparatus. Nucleus.
AhECT1b1Z1C0T.1Chr16125,362,256125,366,581+65671,736.698.47Cell membrane. Nucleus.
AhECT7a7B7L4H.1Chr0199,992,04499,997,188+64671,727.276.02Cell membrane. Chloroplast.
Nucleus.
AhECT8a81NSYN.1Chr0745,863,40345,868,545−58964,907.376.92Nucleus.
AhECT8b8B5AYJ.1Chr18103,869,388103,874,516−58964,834.196.95Nucleus.
AhECT4b8R4GHU.1Chr13128,662,389128,666,999+68975,301.506.72Nucleus.
AhCPSF30a9EB9RH.1Chr048,598,0548,605,707+69676,273.836.43Nucleus.
AhECT7dCFVF1U.1Chr1413,427,39113,432,665+66172,595.876.84Cell wall.
Chloroplast.
Nucleus.
AhECT1aFB9VDL.1Chr0694,533,55594,537,885+65671,762.808.50Nucleus.
AhECT7cK8ASES.1Chr11144,173,624144,178,221−82290,990.616.54Chloroplast.
Nucleus.
AhECT4aL35IVM.1Chr03124,995,564125,000,166+69075,397.666.72Nucleus.
AhECT11bMCG1UL.1Chr144,350,6894,355,084+52257,954.258.42Nucleus.
AhECT12aNNP0DQ.1Chr0513,002,48513,006,368+33838,032.856.63Nucleus.
AhECT5bQH21S6.1Chr14509,475515,570+65471,352.465.64Nucleus.
AhECT12bTXC2DL.1Chr1513,648,20313,652,147+29833,666.506.41Nucleus.
AhECT5aU8THEZ.1Chr04104,936111,353+65471,407.535.66Nucleus.
AhECT11aWRM861.1Chr043,042,0963,047,064+50155,792.098.71Nucleus.
AhCPSF30bXVC3D4.1Chr1410,083,14010,090,746+69676,235.816.21Nucleus.
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Wang, W.; Bian, J.; Liu, X.; Liu, X. Characterization of the N6-Methyladenosine Gene Family in Peanuts and Its Role in Abiotic Stress. Int. J. Plant Biol. 2025, 16, 7. https://doi.org/10.3390/ijpb16010007

AMA Style

Wang W, Bian J, Liu X, Liu X. Characterization of the N6-Methyladenosine Gene Family in Peanuts and Its Role in Abiotic Stress. International Journal of Plant Biology. 2025; 16(1):7. https://doi.org/10.3390/ijpb16010007

Chicago/Turabian Style

Wang, Wei, Jianxin Bian, Xiaoyu Liu, and Xiaoqin Liu. 2025. "Characterization of the N6-Methyladenosine Gene Family in Peanuts and Its Role in Abiotic Stress" International Journal of Plant Biology 16, no. 1: 7. https://doi.org/10.3390/ijpb16010007

APA Style

Wang, W., Bian, J., Liu, X., & Liu, X. (2025). Characterization of the N6-Methyladenosine Gene Family in Peanuts and Its Role in Abiotic Stress. International Journal of Plant Biology, 16(1), 7. https://doi.org/10.3390/ijpb16010007

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