Genome-Wide Characterization of the ALKBH Gene Family Reveals a Potential Role of PgALKBH10 in Multiple Abiotic Stress Responses in Panax ginseng C. A. Mey.

Abstract

Background/Objectives: N6-methyladenosine (m⁶A) is a prevalent RNA modification that significantly influences various biological processes. AlkB homologs (ALKBHs) belong to the family of specific demethylases and, by regulating m⁶A methylation, are known to be involved in the modulation of plant stress responses. However, the ALKBH gene family has not been systematically characterized in ginseng. Methods: A genome-wide identification and characterization of the ALKBH gene family in ginseng were performed using a telomere-to-telomere reference genome. Phylogenetic relationships, gene structures, conserved motifs, 3D structures, chromosomal distribution, syntenic relationships, cis-acting regulatory elements, protein-protein interaction (PPI) networks, and expression profiles were analyzed. Transcriptome datasets covering multiple tissues, developmental stages, cultivars, and abiotic stress treatments were examined. Candidate stress-responsive genes were further validated by qRT-PCR. Results: A total of 17 PgALKBH genes were identified and classified into seven subfamilies. Structural analyses revealed conserved motifs, exon–intron organization, and 3D structures among members within the same subfamily. Chromosomal localization and synteny analyses suggested that the PgALKBH family has been evolutionarily conserved between ginseng and Arabidopsis and has primarily undergone purifying selection during its expansion. Promoter analysis identified abundant light-, hormone-, and stress-responsive cis-elements. Expression profiling revealed distinct tissue- and developmental stage-specific patterns. The PPI analysis suggested that PgALKBH proteins, especially PgALKBH10, may play a central role in m⁶A-mediated RNA regulation in ginseng. Transcriptome and qRT-PCR analyses further showed that PgALKBH genes respond differentially to drought, cold, and salt stresses. Notably, PgALKBH10 was induced under all three stress conditions. Conclusions: This study provides a comprehensive characterization of the ALKBH gene family in ginseng and identifies PgALKBH10 as a promising candidate involved in multiple abiotic stress responses. These findings establish a foundation for elucidating the roles of RNA m⁶A demethylation in ginseng and provide valuable genetic resources for developing stress-tolerant ginseng cultivars.

1. Introduction

RNA modifications play a vital role in defining RNA characteristics and functions by influencing its stability, translation, splicing, and transport [1,2]. More than 170 types of RNA modification have been identified [3]. Within the eukaryotic epitranscriptome, N6-methyladenosine (m⁶A) stands out as an exceptionally extensively studied chemical modification of RNA. In RNA molecules, methyltransferases catalyze the methylation of adenosine (A) by substituting the hydrogen atom at the nitrogen-6 (N6) position with a methyl group (CH3) [4]. In plants, m⁶A methylation levels on messenger RNAs (mRNAs) are controlled by three main groups of proteins: readers, writers, and erasers [5]. The writer complex, responsible for m⁶A deposition, consists of methyltransferases including MTA, MTB, FKBP12 interacting protein 37 (FIP37), as well as VIRILIZER and HAKAI [6,7,8,9]. Proteins containing YTH domains, including ECT2-4 and CPSF30-L, act as m⁶A readers by selectively recognizing methylated transcripts and mediating downstream regulatory processes. This specific binding mechanism is fundamental for the control of gene expression and the orchestration of diverse biological processes [10,11]. AlkB homologs (ALKBH) are Fe²⁺ and α-ketoglutarate-dependent dioxygenases that act as specific demethylases. They catalyze the removal of methyl groups from various substrates, including proteins, DNA, and RNA through hydroxylation. The substrate specificity and biological functions of different ALKBH family members are influenced by structural variations that affect how they recognize and interact with their targets [12,13].

The alkB gene was first identified in Escherichia coli in 1983 as a regulator of sensitivity to methyl methane sulfonate [14]. Ever since then, ALKBHs have been widely identified across diverse organisms and are increasingly recognized for their roles in RNA epigenetic regulation. In plants, ALKBH-mediated m⁶A demethylation has been implicated in multiple developmental processes. For example, in Arabidopsis, ALKBH10B regulates m⁶A methylation to control the expression of FLOWERING LOCUS T (FT), SQUAMOSA PROMOTER BINDING PROTEIN LIKE 3 (SPL3), and SPL9, thus playing a key role in the floral transition. Plants carrying the non-functional alkbh10b allele exhibit a significant delay in flowering onset and impaired vegetative growth [12]. FvALKBH10B operates within a complex regulatory cascade involving the abscisic acid (ABA) signaling pathway, where ABA-Responsive Element Binding Factor 3 (FvABF3) induces its expression in strawberries. The mutation of FvALKBH10B results in delayed fruit ripening and widespread m⁶A hypermethylation, which impacts numerous genes central to ripening processes. By stabilizing the mRNA of SEPALLATA3 (FvSEP3) through demethylation, FvALKBH10B indirectly influences a network of ripening-related genes, including those involved in ABA and anthocyanin biosynthesis [15]. Beyond developmental regulation, ALKBH proteins also play critical roles in plant responses to abiotic stress. The alkbh10b mutants of Arabidopsis showed increased sensitivity to drought stress, whereas plants overexpressing ALKBH10B demonstrated improved drought tolerance. Several m⁶A-modified transcripts associated with the drought stress response exhibited elevated m⁶A levels in the alkbh10b mutants. Under dehydration stress, the decay rates of transcripts modified by m⁶A were higher in alkbh10b mutants as opposed to the wild type [16]. Similarly, in cotton, GhALKBH10B regulates essential drought adaptation networks by lowering the m⁶A levels and mRNA stability of genes related to the ABA and calcium signaling pathways [17]. Moreover, GhALKBH10 modulates salt stress response genes via the m⁶A demethylation pathway. Silencing GhALKBH10 leads to elevated expression of salt stress-related genes, including GhSYTA (Synaptotagmin A), GhMDH (chloroplast malate dehydrogenase), and GhCIPK6 [18].

Within the pharmacopoeia of traditional Chinese medicine, ginseng is renowned as a botanical of exceptional value and therapeutic importance. The therapeutic value of ginseng is largely attributed to its secondary metabolites, particularly ginsenosides, which exert potent anticancer, anti-inflammatory, and immunomodulatory effects [19,20]. The accumulation of secondary metabolites, as well as overall plant growth and productivity, is strongly influenced by environmental conditions, including abiotic stresses [21,22]. Emerging evidence suggests that epigenetic regulation, particularly m⁶A modification, may play a crucial role in integrating stress signals with metabolic and developmental processes [23,24]. Therefore, elucidating the function of RNA demethylases such as ALKBH proteins in ginseng is of particular importance for understanding the molecular mechanisms underlying both stress adaptation and the formation of medicinal quality traits.

Despite the recognized importance of ALKBH proteins in other plant species, the roles of the ALKBH gene family in ginseng remain largely unknown, particularly regarding their involvement in responses to abiotic stress conditions. The recently assembled telomere-to-telomere (T2T) reference genome of ginseng enabled unprecedented assembly continuity and completeness, providing a more reliable genomic framework for the identification and characterization of the ALKBH gene family [25]. This research identified 17 potential PgALKBH genes within the ginseng genome. We then performed an in-depth characterization, which included their phylogenetic history, genomic localization, exon-intron organization, and promoter-associated cis-regulatory elements. In addition, the expression patterns of these PgALKBH genes were explored across a range of tissues and organs, as well as in roots at four unique annual growth stages and from 42 cultivated ginseng varieties. Furthermore, the expression of PgALKBH genes under different abiotic stresses was verified by Quantitative Real-Time PCR (qRT-PCR). Collectively, this study lays a solid foundation for further investigation into the potential roles of PgALKBH genes in abiotic stress adaptation in ginseng.

2. Materials and Methods

2.1. Identification and Analysis of the Physicochemical Properties of ALKBH Genes in Ginseng

The source data for this work, comprising the T2T reference genome and its gene annotations for a China ginseng accession, were obtained as described in a previous report that documented their public release [25]. The TAIR database (https://www.arabidopsis.org/) served as the primary source for collecting all amino acid sequences corresponding to the Arabidopsis ALKBH gene family [26]. Candidate ALKBH genes were identified by aligning these protein sequences against the ginseng protein sequences with the blastp algorithm. The 2OG_FeII_Oxy_2 domain (PF13532) within candidate ALKBH genes was confirmed via analyzing with the NCBI Conserved Domain Database (CDD) [27]. Further validation of the identified ALKBH genes was performed using HMMER [28]. The chromosomal coordinates for each PgALKBH gene were retrieved from the annotation files. This locational information was then analyzed with the Toolkit for Biologists integrating various biological data-handling tools (TBtools v2.486) program [29]. The ExPASy proteomics server was employed to determine the fundamental physicochemical properties, specifically the molecular weight (MW) and theoretical isoelectric point (pI) of the ginseng ALKBH proteins [30].

2.2. Phylogenetic, Gene Structure, and Conserved Motif Analysis of PgALKBHs

A multiple alignment was produced by aligning the ALKBH protein sequences derived from ginseng, Arabidopsis, and wheat. The alignment itself was performed with the MUSCLE algorithm as implemented in MEGA11. The evolutionary relationships were inferred through a phylogenetic tree generated in MEGA11 with the neighbor-joining (NJ) method (Poisson model, 500 bootstraps), which was subsequently rendered and polished using Evolview v2 (https://www.evolgenius.info/evolview-v2/ (accessed on 10 November 2025)). A synteny analysis of the ALKBH genes in ginseng was conducted with TBtools v2.486. Furthermore, the MEME program v5.5.8 (https://meme-suite.org/meme/meme_5.5.8/ (accessed on 10 November 2025)) was employed to detect and analyze conserved protein motifs present in the ginseng ALKBH family. The TBtools v2.486 platform was also utilized to generate a visual illustration of the architectural features for each gene.

2.3. 3D Structure Prediction of PgALKBH Protein

The three-dimensional (3D) structures of the ginseng PgALKBH proteins were predicted using the AlphaFold Protein Structure Database (https://alphafold.com/) [31]. The resulting structural models were then visualized using PyMOL (version 3.1.6).

2.4. Chromosomal Distribution and Synteny Analysis of PgALKBHs

The physical positions of the ginseng ALKBH genes on the chromosomes were ascertained by referencing the annotation data of the reference genome from Jilin Province, China [25]. TBtools v2.486 was then used to generate a diagram illustrating the distribution of these genes from top to bottom based on their genomic positions. The genome sequence and annotation files of Arabidopsis were obtained from the public genomic database Ensembl Plants (https://plants.ensembl.org/index.html (accessed on 10 November 2025)). Bidirectional BLASTP comparisons were conducted using the One Step MCScanX Wrapper in TBtools v2.486 and the resulting synteny file was visualized using the Dual Synteny Plot module in TBtools v2.486.

2.5. Identification of Regulatory Motifs in the PgALKBH Gene Promoters

To identify potential cis-regulatory elements, the 2000 bp sequences upstream of each ALKBH gene’s coding region were first obtained with TBtools v2.486 and defined as the promoters. These promoter sequences were then submitted to the PlantCARE server (https://bioinformatics.psb.ugent.be/webtools/plantcare/html/ (accessed on 10 November 2025)) for motif scanning and prediction [32]. The spatial organization and layout of these elements were afterwards graphically depicted by employing the functionalities of TBtools v2.486.

2.6. Protein-Protein Interaction Network Analysis

The protein-protein interaction (PPI) network of PgALKBH proteins and their interacting partners was predicted using TBtools v2.486, based on representative Arabidopsis protein sequences. The resulting network was visualized and analyzed in Cytoscape (version 3.10.3) to identify highly interconnected PgALKBH-associated modules within the network [33].

2.7. Expression Profiles Analysis of PgALKBHs

Publicly available RNA-Seq datasets (NCBI accession: PRJNA302556), collected from Jilin, China, were utilized for this analysis. The data comprised a wide array of samples, including materials from 14 different ginseng tissues, four distinct age groups (5, 12, 18, and 25 years), and root samples from 42 unique four-year-old cultivars (Table S1). In addition, RNA-Seq datasets associated with abiotic stress treatments were retrieved from the Ginseng Genome Data Resource “http://ginsengdb.snu.ac.kr (accessed on 10 November 2025)”.

Raw sequencing data were first subjected to quality assessment, and adapter sequences as well as low-quality reads were removed using fastp to obtain high-quality clean reads [34]. The reference genome was indexed using HISAT2, and the quality-filtered paired-end clean reads were aligned to the reference genome [35]. The resulting alignments were assembled into transcripts using StringTie, and gene- and transcript-level expression abundances were calculated as transcripts per million (TPM) [36]. In addition, transcript abundance was independently quantified using the pseudo-alignment algorithm implemented in Kallisto to validate the quantification results generated by StringTie [37]. Differentially expressed genes (DEGs) under different stress conditions were identified using DESeq2 [38], with the selection criteria of log₂FoldChange > 1 and an adjusted p-value < 0.01.

We explored the correlations between the PgALKBH genes and cold-, salt-, and drought-stress-related genes in ginseng through correlation analysis. Correlation coefficients were calculated using Origin 2021, and the significance level was judged by the p-value (p < 0.05).

2.8. Plant Materials and Stress Treatments

The Jilin ginseng cultivar “Fuxing No. 2”, sourced from Jilin Province, China, was utilized to generate the adventitious roots used in this study. The plant materials were housed in the Medical Laboratory Testing Technology and Analytical Laboratory of Beihua University.

For drought stress treatments, adventitious roots (cultured for 25 days) were exposed to B5 medium containing 5% PEG 6000. Samples were collected at 0, 3, 6, 9, 12, and 15 days post-treatment. For cold treatment, adventitious roots cultured on B5 medium for 25 days were transferred to 4 °C. Samples were collected at 0, 6, 12, 24, 48, and 72 h post-treatment. For the salt treatment, adventitious roots of uniform size (~1 cm) were placed on B5 medium supplemented with different concentrations of NaCl (0, 70, 80, 90, and 100 mM) and cultured at 25 °C for 30 days. At the conclusion of the experimental treatments, all samples were rapidly frozen using liquid nitrogen and were subsequently held at −80 °C for future gene expression studies.

2.9. qRT-PCR Analysis

Total RNA was extracted from the “Fuxing No. 2” variety of Jilin ginseng through the TRIzol protocol. Subsequently, cDNA was synthesized from the purified RNA with the Super RT III Kit provided by Biosharp Biotech (Labgic Tech-nology Co., Ltd., Beijing, China). Relative transcript abundance was then quantified by qRT-PCR, for which all primers were obtained from Sangon Biotech (Shanghai, China) and are listed in Table S2. The β-actin gene was utilized as the endogenous reference for all assays. The qRT-PCR reactions themselves were prepared using the SYBR Premix Ex Taq™ II (Tli RNaseH Plus) kit (Takara Biomedical Technology (Beijing) Co., Ltd., Beijing, China). To ensure reliability and reproducibility, qRT-PCR analyses were performed using three independent biological replicates. Relative gene expression levels were calculated using the 2^−ΔΔCT method. Statistical analyses and graphical visualizations were performed using GraphPad Prism version 10.1 (GraphPad Software, San Diego, CA, USA). Differences between two groups were evaluated using a two-tailed Student’s t-test. Differences were considered statistically significant at p < 0.05 and highly significant at p < 0.01. Because only pairwise comparisons were performed, no multiple-comparison correction was applied [39].

3. Results

3.1. Identification and Characterization of ALKBH Genes in Ginseng

A total of 17 ALKBH genes were identified in ginseng (Table 1). The molecular features and protein properties of these ALKBH genes were analyzed, including gene IDs, number of amino acids (aa), molecular weights (MW), isoelectric points (pI), chromosomal locations, and predicted subcellular localizations. The lengths of the encoded ALKBH proteins ranged from 217 aa (PgALKBH7) to 612 aa (PgALKBH10). Their molecular weights spanned approximately from 24.4 kDa (PgALKBH7) to 67.9 kDa (PgALKBH10). The pI values varied between 4.63 (PgALKBH8B) and 9.51 (PgALKBH2B). Subcellular localization predictions showed that 3 proteins (PgALKBH1D-2, PgALKBH9B-1, and PgALKBH9B-2) were localized in the cytoplasm, and PgALKBH1D-3 was localized in the plastid. Furthermore, a nuclear destination was predicted for the 13 other ALKBH proteins in the set.

Table 1.

Protein characteristics of predicted PgALKBH genes in ginseng.

Gene Name	Gene ID	Number of Amino Acid	Molecular Weight (MW)	Isoelectric Point (pI)	Location	Subcellular Localization
PgALKBH1A-1	pg_1000211.t01	360	40,670.4	5.9	Chr01:1796529-1800141(−)	Nucleus
PgALKBH1A-2	pg_9011448.t01	330	37,376.71	6.04	Chr09:146692600-146695997(−)	Nucleus
PgALKBH1D-1	pg_3013481.t01	300	33,350.11	7.63	Chr03:175959207-175962520(+)	Nucleus
PgALKBH1D-2	pg_4011596.t01	362	40,924.44	8.51	Chr04:170672519-170675042(−)	Cytoplasm
PgALKBH1D-3	pg_10011064.t01	387	43,619.81	9.47	Chr10:144323551-144326425(−)	Plastid
PgALKBH1D-4	pg_19000070.t02	408	45,197.03	8.66	Chr19:829595-834230(−)	Nucleus
PgALKBH2A	pg_23000155.t01	244	27,972.02	8.97	Chr23:1456106-1458537(−)	Nucleus
PgALKBH2B	pg_24007950.t01	241	27,920.07	9.51	Chr24:104287641-104291106(+)	Nucleus
PgALKBH6	pg_14000020.t01	258	29,141.97	6.3	Chr14:266182-272568(+)	Nucleus
PgALKBH7	pg_20002311.t02	217	24,436.63	7.1	Chr20:37126701-37128330(−)	Nucleus
PgALKBH8A	pg_4000687.t01	265	30,477.21	4.83	Chr04:6798400-6803653(−)	Nucleus
PgALKBH8B	pg_10000671.t01	267	30,693.46	4.63	Chr10:5711525-5716627(−)	Nucleus
PgALKBH9A-1	pg_6001170.t01	508	57,094.53	6.66	Chr06:11396976-11401426(−)	Nucleus
PgALKBH9A-2	pg_22001132.t01	508	57,116.48	6.11	Chr22:9624431-9628852(−)	Nucleus
PgALKBH9B-1	pg_8011724.t01	472	52,788.3	8.67	Chr08:158383631-158389060(+)	Cytoplasm
PgALKBH9B-2	pg_15008195.t01	560	62,582.53	8.94	Chr15:111006245-111013244(+)	Cytoplasm
PgALKBH10	pg_5000707.t01	612	67,908.19	6.7	Chr05:6197201-6203803(−)	Nucleus

3.2. Phylogenetic Analysis of ALKBH Genes

To elucidate the evolutionary divergence and provide a classification for the ALKBH gene family in ginseng, a phylogenetic analysis was undertaken. The tree itself was built based on aligned protein sequences from ginseng alongside those from both Arabidopsis and wheat. The PgALKBH gene family was divided into seven subfamilies, which are generally categorized according to their homology with Arabidopsis ALKBH proteins. Six PgALKBH genes (PgALKBH1A-1, PgALKBH1A-2, PgALKBH1D-1, PgALKBH1D-2, PgALKBH1D-3, and PgALKBH1D-4) were classified into the ALKBH1 subfamily. Four PgALKBH genes (PgALKBH9A-1, PgALKBH9A-2, PgALKBH9B-1, and PgALKBH9B-2) belonged to the ALKBH9 subfamily. The ALKBH2 and ALKBH8 subfamilies each included two PgALKBH genes. PgALKBH6, PgALKBH7, and PgALKBH10 were assigned to the ALKBH6, ALKBH7, and ALKBH10 subfamilies, respectively. Notably, PgALKBH10 may serve as a candidate m⁶A RNA demethylase due to its close evolutionary relationship with the well-characterized AtALKBH10B, which is known for removing m⁶A modifications from RNA (Figure 1).

Figure 1.

A phylogenetic tree was generated to elucidate the evolutionary relationships of the ALKBH protein family among ginseng, Arabidopsis, and wheat. The tree’s construction was based on the NJ algorithm, and statistical confidence for the nodes was assessed via 500 bootstrap iterations. The resulting phylogenetic tree segregated the ALKBH genes of ginseng into a total of seven clades. Pg, ginseng; At, Arabidopsis thaliana; Ta, wheat.

3.3. Structural and Architectural Characterization of the PgALKBHs

To identify conserved protein motifs, the ALKBH sequences were submitted to the MEME online tool. This analysis revealed a total of ten distinct motifs that are broadly shared across the ALKBH protein family. The gene structures were characterized in a separate analysis. All PgALKBH proteins contained motif 1 and motif 2, except PgALKBH1A-2. ALKBH proteins that group together within the same subfamily also exhibit comparable motif compositions, which suggests a degree of functional overlap among these members. Motifs 3, 5, and 7 were unique to the PgALKBH proteins (PgALKBH9A-1, PgALKBH9A-2, PgALKBH9B-1, PgALKBH9B-2, PgALKBH10) assigned to the ALKBH9 and ALKBH10 subfamilies (Figure 2A). The presence of the 2OG_FeII_Oxy_2 domain in the PgALKBH proteins was confirmed. In most ALKBH proteins, the 2OG_FeII_Oxy_2 domains are located close to the C-terminal region (Figure 2B). The presence of an identical conserved domain among these PgALKBH proteins suggested potential functional redundancy.

Figure 2.

Comprehensive analysis of the ginseng ALKBH gene family. This includes an examination of their phylogenetic relationships, conserved protein motifs, domain architecture, and exon-intron organization. (A) Evolutionary relationships and conserved motif patterns within the PgALKBH protein family. Each colored box corresponds to a distinct motif. (B) Conserved domain of ALKBH proteins in ginseng. (C) Gene structures for the PgALKBH family. In this schematic, yellow boxes represent the exons, while the intervening introns are indicated by black lines. The untranslated regions (UTRs) are shown as green boxes.

Structural variation was observed across the ginseng ALKBH gene family, as determined by an analysis of their exon-intron layouts. The resulting exon count for these genes was found to vary between three and nine. The genes within each subfamily exhibited similar structural characteristics (Figure 2C). A notable structural intricacy and variety was observed among the candidate genes across the various subfamilies. This finding implies that the PgALKBH genes in ginseng have probably retained their primary functions but could have also evolved to perform new biological tasks.

3.4. 3D Structure Analysis of OsALKBH Proteins

To further investigate the structural characteristics of the PgALKBH proteins, their 3D structures were predicted using AlphaFold (Figure 3). All 17 PgALKBH proteins exhibited well-defined tertiary structures composed predominantly of α-helices and β-sheets. Proteins belonging to the same subfamily displayed highly similar overall folding patterns. For example, PgALKBH1A-1 and PgALKBH1A-2 exhibited nearly identical structural architectures, as did PgALKBH9A-1 and PgALKBH9A-2, as well as PgALKBH9B-1 and PgALKBH9B-2, indicating a high degree of structural conservation following gene duplication. Structural variations were observed among different PgALKBH subfamilies, suggesting potential functional divergence among these proteins. Despite sharing conserved domain architectures, the distinct structural features may contribute to their diverse biological functions.

Figure 3.

3D structural models of PgALKBH proteins. α-helices, β-sheets, and random coils are represented by blue helices, yellow arrows, and wheat lines, respectively.

3.5. Genomic Localization and Synteny Analysis of the PgALKBHs

Ginseng is known to be an allotetraploid, and earlier studies consequently partitioned its chromosome set into subgenome A and subgenome B. The physical positions of the ginseng ALKBH genes within the genome were ascertained by conducting a search with the BLASTN program. The PgALKBH genes were approximately evenly distributed between the two subgenomes, with 8 genes anchored on subgenome A and 9 genes on subgenome B. Chromosomes 4 and 10 each harbor two PgALKBH genes, while chromosomes 1, 3, 5, 6, 8, 9, 14, 15, 19, 20, 22, 23, and 24 each contain a single PgALKBH gene. Eight PgALKBH genes are located on corresponding chromosomes across subgenomes A and B (Figure 4A). All PgALKBH genes are positioned near the terminal regions of the chromosomes, except for PgALKBH7. These results indicated that ginseng’s evolutionary history has likely been influenced by diverse evolutionary trajectories.

Figure 4.

Genomic distribution and synteny analysis of the ginseng ALKBH gene family. (A) Chromosomal localization of PgALKBHs across both subgenomes A and B. (B) Synteny analysis of ALKBH genes between ginseng and Arabidopsis. The blue lines indicate syntenic ALKBH gene pairs. (C) Collinearity analysis of PgALKBHs in ginseng chromosomes. The red lines indicate collinear pairs of PgALKBH family genes.

To investigate the evolutionary conservation of the PgALKBH gene family, a comparative synteny analysis was performed between ginseng and Arabidopsis (Figure 4B). A total of 12 orthologous gene pairs involving nine PgALKBH genes were identified between the two species (Table S3). These included PgALKBH1D-1, PgALKBH1D-2, PgALKBH1D-3, PgALKBH1D-4, PgALKBH8A, PgALKBH8B, PgALKBH9A-1, PgALKBH9A-2, and PgALKBH10. Among these, PgALKBH9A-1 and PgALKBH9A-2 each exhibited syntenic relationships with two Arabidopsis ALKBH genes (AtALKBH9B and AtALKBH9C), while PgALKBH10 showed synteny with AtALKBH10A and AtALKBH10B, indicating that these genes have retained conserved genomic relationships during evolution. In addition, members of the PgALKBH1D subfamily displayed conserved collinearity with AtALKBH1B and AtALKBH1D, whereas PgALKBH8A and PgALKBH8B were syntenic with AtALKBH8B. Overall, the conserved syntenic relationships between ginseng and Arabidopsis suggest that several PgALKBH genes have been evolutionarily conserved following species divergence.

An intra-specific collinearity analysis of the ginseng genome was conducted to understand the evolutionary expansion of the PgALKBH gene family, resulting in the identification of seven distinct pairs of syntenic PgALKBH genes. Most of these gene pairs are located on corresponding chromosomes within the subgenomes A and B, except for PgALKBH2A and PgALKBH2B (Figure 4C). The selection forces on duplicated PgALKBH genes were determined by analyzing their non-synonymous (Ka) and synonymous (Ks) substitution rates, along with the Ka/Ks ratio. The calculated Ka/Ks values for every gene pair were found to be lower than 1, falling within the range of 0.265 to 0.953 (Table S4). This outcome suggested that the evolution of the PgALKBH gene family has been mainly governed by purifying selection.

3.6. Identification of Putative Cis-Acting Regulatory Elements in PgALKBH Gene Promoters

Understanding the cis-regulatory elements that control the transcriptional regulation of PgALKBH genes is essential for uncovering their biological roles in ginseng. An analysis of the PgALKBH genes revealed 386 cis-acting elements corresponding to nineteen distinct functional groups (Figure 5A). These were then classified into five principal types: those responsive to light, hormones, developmental cues, and stress, along with a category for other miscellaneous elements. A total of 192 light-responsive elements were identified, representing the largest category of regulatory elements and occurring in all PgALKBH gene promoters (Table S5). A considerable number of motifs involved in stress signaling were also found. More than 60% of these were drought-responsive MYB binding sites (MBS) and anaerobic induction elements, highlighting their importance in stress responses (Figure 5B). A number of hormone-responsive regulatory loci were found, including those that respond to methyl jasmonate (MeJA), ABA, gibberellin (GA), auxin, and salicylic acid (SA). The presence and arrangement of cis-acting elements in promoter regions suggest that MeJA is the dominant regulator of PgALKBH genes (Figure 5C). Moreover, the analysis also identified cis-acting elements that are involved in developmental processes and various other roles (Figure 5A). The abundance of light-, ABA-, MeJA-, and anaerobic-responsive elements in PgALKBH promoters suggested that their expression is regulated by light, phytohormones, and environmental stresses, which likely influence ginseng development.

Figure 5.

Analysis of predicted cis-acting regulatory motifs in the PgALKBH gene family. (A) Visualization of the layout for predicted cis-elements within the promoter sequences of each PgALKBH gene. In the diagram, the gray lines signify the upstream sequences. Specific cis-acting motifs are indicated by colored squares, with a corresponding legend provided on the right. (B) The abundance and types of predicted cis-elements related to various stress responses. (C) The abundance and categories of identified cis-elements associated with phytohormone signaling.

3.7. PPI Network of PgALKBH Proteins

To explore the potential functions of PgALKBH proteins, a PPI network was predicted based on Arabidopsis orthologs using TBtools and visualized with Cytoscape (Figure 6). PgALKBH10 was identified as the central hub with the highest connectivity, followed by PgALKBH7, PgALKBH2A, PgALKBH2B, PgALKBH9A-1/2, PgALKBH1D-2, and PgALKBH1D-3, whereas PgALKBH1D-1 and PgALKBH1D-4 were peripheral nodes with fewer interactions.

Figure 6.

Analysis of PPI network of PgALKBH proteins. The darker the color, the higher the node degree.

Functional annotation revealed that the interacting partners encompassed core components of the m⁶A regulatory system (Table S6). These included m⁶A writers, represented by N6-adenosine-methyltransferase MT-A70-like proteins pg_17000069 and pg_3000025, and m⁶A readers, represented by the two YTH-domain proteins PgYTH11 and PgYTH12 that were previously characterized by our group [40]. Together with the PgALKBH proteins themselves, which function as m⁶A erasers, the network thus encompassed all three core components of the m⁶A regulatory system. Several uncharacterized proteins were also present as potential novel interactors. The co-occurrence of m⁶A writers, readers, and erasers within a single PgALKBH-centered network suggests that PgALKBH proteins, particularly the hub protein PgALKBH10, may participate in m⁶A-mediated RNA regulation in ginseng through coordinated interactions with these functional partners.

3.8. Transcriptional Profiling of the Ginseng ALKBH Gene Family

To characterize the transcriptional profiles of the PgALKBH gene family in ginseng, we analyzed expression data derived from a diverse set of samples. This dataset included root tissues from four different growth years (5, 12, 18, and 25), a panel of 42 agricultural cultivars (S1–S42), and fourteen different plant tissues (Table S7). Among all PgALKBH genes, PgALKBH1D-4 exhibited consistently high expression levels in roots across the four age groups, with TPM values of 70.35 (5-year-old), 26.12 (12-year-old), 57.27 (18-year-old), and 22.37 (25-year-old), which were generally higher than those of other family members, indicating that PgALKBH1D-4 may be the predominantly expressed member of the PgALKBH gene family throughout root development. The expression level of PgALKBH6 in 25-year-old roots was higher than in roots of the other ages, with TPM values of 24.65 (Figure 7A). Among the 17 ALKBH genes, PgALKBH6 stood out for its markedly high expression levels across most of the fourteen tested ginseng tissues, with the notable exception of the leaf blade. This contrasts sharply with the remaining fourteen family members, which all demonstrated comparatively low transcriptional activity in these same tissues. This suggested that PgALKBH6 may have distinct biological functions and regulatory roles in ginseng. The PgALKBH9B-1 gene showed higher expression levels in leg root and fiber root, with TPM values of 27.50 and 23.30, respectively, than in other tissues (Figure 7B). Across 42 different Panax ginseng varieties, PgALKBH9B-1, PgALKBH6, PgALKBH9B-2, and PgALKBH1D-4 exhibited higher expression levels than the other PgALKBH genes. In particular, PgALKBH9B-1 displayed relatively high expression levels across all 42 varieties (Figure 7C).

Figure 7.

Expression patterns of PgALKBHs. (A) Transcriptional profiling of PgALKBH genes in ginseng roots across different growth stages (5, 12, 18, and 25 years). (B) Expression levels within fourteen distinct tissues from a four-year-old ginseng plant. These samples included the seed, fruit flesh, fruit pedicel, fruit peduncle, leaf blade, leaflet pedicel, leaf peduncle, stem, arm root, rhizome, main root cortex, main root epidermis, leg root, and fiber root. (C) Expression profiles of PgALKBH genes in roots from 42 different farm cultivars of 4-year-old ginseng.

3.9. Expression Analysis of PgALKBHs Under Abiotic Stress

To characterize the transcriptional profiles of the PgALKBH gene family under abiotic stress in ginseng, we analyzed RNA-Seq datasets from the Ginseng Genome Data Resource. The majority of PgALKBH genes showed no significant changes in expression following stress treatment. PgALKBH10 showed increased expression across the three different stress conditions, particularly under drought stress (Figure 8A).

Figure 8.

Expression profiling of PgALKBH genes under drought, cold and salt stress. (A) Heatmap showing the expression levels of PgALKBH family members under control, drought, cold, and salt stress conditions. (B–D) Expression patterns of PgALKBH10, PgALKBH1D-1, and PgALKBH2B in response to drought treatment over different time points (0, 3, 6, 9, 12, and 15 d). (E,F) Expression dynamics of PgALKBH2B and PgALKBH10 under cold treatment at different time points (0, 6, 12, 24, 48, and 72 h). (G) Expression response of PgALKBH10 to different salt concentrations (0, 70, 80, 90, and 100 mM). “*” indicates p  ≤  0.05; “**” indicates p  ≤  0.01; “ns” indicates not statistically significant.

Based on differential expression analysis of the transcriptome (log₂FoldChange > 1 and adjusted p < 0.01), candidate genes with prominent expression alterations were identified from the PgALKBH family. Subsequent qRT-PCR validation confirmed that these PgALKBH members exhibited distinct, condition-specific transcriptional responses to drought, cold, and salt stresses. PgALKBH10, PgALKBH1D-1, and PgALKBH2B exhibited significantly higher expression levels than the control at nearly all time points under drought treatment (Figure 8B-D). PgALKBH2B showed significant upregulation at 24, 48, and 72 h under cold stress, whereas PgALKBH10 maintained significantly higher expression levels than the control throughout the entire time course (Figure 8E,F). PgALKBH10 displayed a typical dose-dependent response, with no significant alterations in expression at low salt levels (70–80 mM). The expression of PgALKBH10 increased with rising salt concentrations, particularly at 90 mM and 100 mM (Figure 8G). Functional divergence was observed among PgALKBH family members in ginseng. Notably, PgALKBH10 was up-regulated under drought, low-temperature, and salt stress. The most pronounced increase in expression was observed during the late stage of drought and under high-salt treatment, suggesting that PgALKBH10 may play a pivotal role in abiotic stress responses of ginseng.

Given the limited understanding of stress-related genes in ginseng, Pearson correlation analysis was performed to explore the potential associations between PgALKBH genes and annotated stress-responsive genes under drought, cold, and salt stress conditions (Table S8). The results showed that PgALKBH1D-1, PgALKBH2B, and PgALKBH10 exhibited strong correlations (|r| > 0.95) with several stress-related genes (e.g., pg_9010504, pg_15003129, and pg_5002129), suggesting their involvement in multiple stress adaptation processes.

4. Discussion

A wide variety of biological processes are profoundly influenced by the m⁶A modification, which serves a pivotal function in governing the stability and translational efficiency of mRNA transcripts [41]. In mammals, the identification of m⁶A demethylases, primarily ALKBH5 and fat mass and obesity-associated protein (FTO), revealed that m⁶A modification is reversible [42,43]. This discovery has significantly accelerated research on the functional roles of m⁶A in various organisms [44]. More thorough and precise investigations of plant gene families have been made possible by the recent surge in available reference genomes. This wealth of genomic data is a direct result of the swift progress made in whole-genome sequencing technologies. Although no homolog of the FTO gene has been found in plants, the widespread presence of the ALKBH gene family across multiple plant species indicates a conserved mechanism for m⁶A demethylation in the plant kingdom. Previous studies have revealed variable numbers of ALKBH genes in different plants, such as 13 in Arabidopsis [45], 22 in soybean [46], 8 in oriental melon [47], and up to 30 in wheat [48], reflecting possible species-specific expansions or contractions related to their evolutionary histories and functional demands. To date, there has been no documentation of the ALKBH gene family in ginseng.

The present investigation led to the discovery and characterization of 17 distinct genes belonging to the PgALKBH family in Panax ginseng. The newly available T2T reference genome of ginseng was crucial for this analysis, as it provided a more complete and contiguous genomic framework than earlier draft assemblies [25]. This advantage is especially important in ginseng, a complex allotetraploid species, because the T2T genome enabled more reliable gene identification, more accurate chromosomal localization, and better resolution of duplicated and syntenic regions. As a result, our analysis provides a more comprehensive and confident overview of the PgALKBH gene family. The chromosomal distribution of PgALKBH genes in ginseng reveals an approximately equal presence on its two subgenomes, A and B, consistent with its allotetraploid nature [25]. The presence of genes on corresponding chromosomes across both A and B subgenomes further supports the idea of conserved genomic segments retained through ginseng’s complex evolutionary history, shaped by distinct evolutionary trajectories in each subgenome. The ALKBH genes were previously reported to be divided into seven subfamilies, with members generally grouped according to their homology to Arabidopsis ALKBH proteins [45]. Similar to Arabidopsis, most species have more members in the ALKBH1, ALKBH9, and ALKBH10 subfamilies. However, the ALKBH10 subfamily gene is absent in tomato, and there is only one ALKBH10 gene in sweet orange. Other ALKBH subfamilies mostly contain 1–2 genes per subfamily across species, except that Populus has 4 genes in the ALKBH8 subfamily and lacks ALKBH7 subfamily genes [49]; pigeon pea lacks genes in the ALKBH6 and ALKBH7 subfamilies [50]. Wheat has 3 genes each in the ALKBH2, ALKBH6, ALKBH7, and ALKBH8 subfamilies [48], highlighting the impact of polyploidy on ALKBH gene family expansion. Only one gene (PgALKBH10) belonging to the ALKBH10 subfamily was identified in this study, suggesting lineage-specific gene loss or divergence affecting potential regulatory mechanisms mediated by the ALKBH10 subfamily. This may have significant implications for m⁶A-mediated regulatory mechanisms, potentially affecting specific pathways reliant on ALKBH10-mediated demethylation.

The expression patterns of the ALKBH gene family across different plant species indicate a strong conservation of tissue-specific roles, highlighting their potential importance in plant development and function. Huang et al. reported that nearly all CsALKBH genes in sweet orange show high expression in callus, while CsALKBH9A exhibits relatively low expression in roots and leaves [51]. This is similar to the low expression of PgALKBH9A in roots and leaves observed in our study. Zhao et al. found that members of the ALKBH1, ALKBH6, and ALKBH9 subfamilies are highly expressed in Populus leaves [49]. This corresponds with our findings that PgALKBH1D-4, PgALKBH6 and PgALKBH9B-1 also show strong expression in leaves, suggesting these ALKBH members may have conserved roles in regulating leaf function. In cotton, GhALKBH1D was reported to be highly expressed in roots [18], consistent with the high-expression pattern of PgALKBH1D-4 in our study. This result implies a comparable function for the gene in the growth, development, or overall biological activity of roots across various species. In potato and wheat, StALKBH9B, StALKBH10, TaALKBH9B and TaALKBH10 are significantly up-regulated in stem and leaf tissues [48,52]. Likewise, PgALKBH9B-1 and PgALKBH10 in our research also show high expression in stems and leaves, further supporting the notion that ALKBH9B-1 and ALKBH10 genes may have conserved functions in the aerial parts of plants. The conserved expression trends highlight evolutionary pressure to maintain ALKBH-mediated m⁶A regulation in specific tissues, while observed expression differences among species may enable specialized adaptations.

Members of the ALKBH family have emerged as important regulators of plant growth, development, and environmental adaptation through their roles in RNA m⁶A demethylation. Increasing evidence indicates that ALKBH-mediated epitranscriptomic regulation participates in abiotic stress signaling pathways by modulating the expression of stress-responsive genes. In Arabidopsis, the demethylase ALKBH9C confers salt tolerance by regulating the transcription levels of the positive effectors SOS1, SAD1, PIP1D and the negative factor PATL1 [53]. In tomato, knocking out SlALKBH9B increased flower drop, whereas overexpressing SlALKBH9B delayed the onset of flower drop. m⁶A modification inhibits drought-induced flower drop by regulating the ethylene synthesis pathway [54]. A study on sugar beet found that salt stress induced diverse expression of BvALKBH genes in its leaves, with BvALKBH10B being significantly upregulated while BvALKBH9B was strongly repressed [55]. Research on potato revealed that several StALKBH family members, including StALKBH9B and StALKBH10B, were significantly upregulated under salt stress but generally downregulated during cold treatment, indicating that the same genes may be differentially regulated under varying stress conditions [52]. Similarly, a study on soybean showed that different genes within the GmALKBH10 subfamily responded to cold, alkaline, and drought stresses with varying intensities and timing. Moreover, the expression peaks of different GmALKBH10 genes after cold treatment occurred at different time points, suggesting their distinct roles during early, middle, and late phases of stress responses [46].

In the present study, transcriptome analysis and qRT-PCR validation revealed that several PgALKBH genes responded significantly to drought, cold, and salt stresses, indicating their potential involvement in abiotic stress adaptation in ginseng. The differential expression patterns observed among PgALKBH genes indicate substantial functional diversification within the ginseng ALKBH family. Such divergence may enable ginseng to fine-tune epitranscriptomic regulation under varying environmental conditions, thereby improving stress adaptability. Among all examined genes, PgALKBH10 emerged as the most promising candidate involved in abiotic stress responses. In addition, the predicted PPI network identified PgALKBH10 as the central hub of the PgALKBH proteins, while Pearson correlation analysis revealed strong positive correlations between PgALKBH10 and multiple stress-responsive genes. Given the established functions of ALKBH proteins as m⁶A demethylases, PgALKBH10 may regulate the stability, splicing, transport, or translation of stress-responsive transcripts through dynamic RNA methylation changes.

Nevertheless, several limitations of the present study should be acknowledged. Although our study identified PgALKBH10 as a promising candidate involved in abiotic stress responses, these findings are primarily based on expression profiling and bioinformatic analyses and therefore do not provide direct evidence of its biological function. In addition, stress-specific treatment conditions were selected according to commonly used experimental protocols for each type of abiotic stress. Consequently, the drought, cold, and salt stress experiments differed in treatment duration, sampling time points, and salt concentrations, which may limit direct comparisons among different stress responses. Taken together, these limitations indicate that the precise molecular mechanisms underlying the function of PgALKBH10 remain to be elucidated. Future studies involving gene overexpression, knockout or knockdown approaches, together with transcriptome-wide m⁶A profiling and identification of downstream target genes, will be necessary to clarify the molecular mechanisms and regulatory pathways mediated by PgALKBH10. Such investigations will advance our understanding of m⁶A-mediated epitranscriptomic regulation in ginseng stress responses and may provide valuable genetic resources for improving stress tolerance in cultivated ginseng varieties.

5. Conclusions

In this study, we performed the first comprehensive genome-wide characterization of the ALKBH gene family in ginseng using the T2T reference genome. A total of 17 PgALKBH genes were identified and systematically characterized through analyses of phylogenetic relationships, gene structures, conserved motifs, chromosomal distribution, syntenic relationships, cis-acting regulatory elements, 3D protein structures, PPI networks, and expression profiles. The results showed that the PgALKBH gene family is highly conserved during evolution while exhibiting evidence of functional diversification. PgALKBH10 was identified as a promising candidate associated with abiotic stress responses. However, its biological function remains to be experimentally validated. Overall, this study provides a comprehensive resource for understanding the evolution and potential functions of the PgALKBH gene family in ginseng and establishes a solid foundation for future functional studies on m⁶A-mediated epigenetic regulation and the molecular breeding of stress-tolerant ginseng cultivars.

Supplementary Materials

The following supporting information can be downloaded at: https://www.mdpi.com/article/10.3390/genes17070793/s1, Table S1: Plant materials used for this study; Table S2: Primers used for qRT-PCR; Table S3: Gene pairs between Arabidopsis and Ginseng; Table S4: The KaKs ratios and estimated divergence times for duplicate pairs of PgALKBHs; Table S5: Cis-acting elements of PgALKBHs; Table S6: PPIs and credibility scores for PgALKBH family members in ginseng; Table S7: The TPM values of 17 PgALKBH genes in 42 ginseng varieties; Table S8: Pearson correlation analysis of PgALKBH genes under drought (|r| > 0.95).

Author Contributions

Y.S.: data curation, investigation, formal analysis, methodology, and writing—original draft. Y.Z.: methodology and writing—original draft. W.Y.: data curation, investigation, writing—review and editing. D.W.: investigation, writing—review and editing. J.H.: writing—original draft preparation. W.H.: funding acquisition, project administration, validation, visualization, writing—review and editing. All authors have read and agreed to the published version of the manuscript.

Institutional Review Board Statement

Not applicable.

Informed Consent Statement

Not applicable.

Data Availability Statement

The original contributions of this study are contained within the article/Supplementary Material.

Conflicts of Interest

The authors declare no conflicts of interest.

Funding Statement

This research was funded by the Science and Technology Development Project of Jilin Province (20240602082RC).