Genome-Wide Identification of the Soybean AlkB Homologue Gene Family and Functional Characterization of GmALKBH10Bs as RNA m6A Demethylases and Expression Patterns under Abiotic Stress

Soybean (Glycine max (L.) Merr) is one of the most important crops worldwide, but its yield is vulnerable to abiotic stresses. In Arabidopsis, the AlkB homologue (ALKBH) family genes plays a crucial role in plant development and stress response. However, the identification and functions of its homologous genes in soybean remain obscured. Here, we identified a total of 22 ALKBH genes in soybean and classified them into seven subfamilies according to phylogenetic analysis. Gene duplication events among the family members and gene structure, conserved domains, and motifs of all candidate genes were analyzed. By comparing the changes in the m6A levels on mRNA from hair roots between soybean seedlings harboring the empty vector and those harboring the GmALKBH10B protein, we demonstrated that all four GmALKBH10B proteins are bona fide m6A RNA demethylases in vivo. Subcellular localization and expression patterns of the GmALKBH10B revealed that they might be functionally redundant. Furthermore, an analysis of cis-elements coupled with gene expression data demonstrated that GmALKBH10B subfamily genes, including GmALKBH10B1, GmALKBH10B2, GmALKBH10B3, and GmALKBH10B4, are likely involved in the cis-elements’ response to various environmental stimuli. In summary, our study is the first to report the genome-wide identification of GmALKBH family genes in soybean and to determine the function of GmALKBH10B proteins as m6A RNA demethylases, providing insights into GmALKBH10B genes in response to abiotic stresses.


Introduction
N6-methyladenosine (m 6 A) is an abundant chemical modification in eukaryotic mRNA that is characterized by dynamic and reversible regulation, and it has a significant impact on RNA function in organisms.Similar to DNA methylation and histone methylation, m 6 A modification requires three types of enzymes including writer, eraser, and reader proteins that dynamically install, remove, and recognize RNA methylation information.In this system, RNA demethylation enzymes are considered to be crucial negative regulators of m 6 A modification.They are capable of recognizing and removing m 6 A modifications in RNA, which are involved in regulating the transcription, splicing, degradation, and translational regulatory processes of RNA [1,2].Alpha-ketoglutaratedependent dioxygenase (AlkB) homologues belong to a specific demethylation family.The members of this family were first identified as DNA repair proteins in E. coli [3].Fat-massand obesity-associated proteins (FTOs) are a m 6 A demethylase found exclusively in mammals [4].The discovery of the m 6 A demethylase FTO indicated that m 6 A modification was stress tolerance in soybean [14][15][16][17][18].For instance, a recent study demonstrated that pseudoresponse regulator 3b (GmPRR3b) negatively regulates the drought response by suppressing the expression of abscisic acid-responsive element-binding factor 3 (GmABF3).Overexpressing GmABF3 can significantly increase the ability to endure drought [14].Additionally, RNA modifications, including m 6 A, play important roles in the response to abiotic stresses.After cadmium treatment, root growth was strongly suppressed in soybean plants.However, the presence of rhizobia can promote root growth and restore the growth performance induced by cadmium stress.These phenotypic alterations could be traced back to the reduction in m 6 A levels on specific genes involved in ROS homeostasis and calcium signaling [15].In the presence of lead, the expression levels of many m 6 A-containing transcripts involved in lead uptake, transport, and accumulation are highly increased in soybean roots, hence enhancing their tolerance to lead accumulation [16].m 6 A RNA modifications also play an important role in the response to light in soybean plants.The core genes in the lightsignaling pathway, such as suppressor of phA-105 1a (GmSPA1a), pseudo-response regulator 5e (GmPRR5e), and blue-light inhibitor of cryptochromes 2b (GmBIC2b), exhibit changes in m 6 A levels on mRNA and in transcript abundance in response to light stimuli [17].However, it remains unclear whether ALKBH family gene-mediated RNA demethylation in soybean is involved in the response to abiotic stress conditions.Therefore, identification and functional analysis of the role of the GmALKBH gene family in stress tolerance are urgently needed.
Here, we identified 22 GmALKBH candidate genes in soybean.The evolutionary relationship, chromosomal location, gene structure, and cis-element regulation of the GmALKBH family were analyzed.Among them, GmALKBH10B1, GmALKBH10B2, GmALKBH10B3, and GmALKBH10B4 were demonstrated to be m 6 A RNA demethylases using an in vivo enzymatic analysis.The tissue-specific expression and subcellular localization data suggest that the expression patterns of the four GmALKBH10B genes varied.In addition, the GmALKBH10B subfamily genes responded differently to various abiotic stresses.Hence, our study provides basic information on the GmALKBH gene family and the expression levels of GmALKBH10Bs upon abiotic stress treatment.

Genome-Wide Identification of the GmALKBH Genes in Soybean
To extensively identify the ALKBH proteins in soybean (Glycine max (L.) Merr.), a BLASTp analysis in the Phytozome V13 database was conducted using the amino acid sequences of the fourteen known Arabidopsis ALKBH family proteins [7,19].As a result, twenty-four homologous proteins from the genome of G. max (Wm82.a4.v1) were obtained.Among them, twenty-two candidates (Table 1) were identified as GmALKBH family members employing the HMM module (Hidden Markov Model) of the AtALKBH protein sequences from Arabidopsis [20,21].To further investigate the evolutionary rela-Plants 2024, 13, 2491 3 of 17 tionship between GmALKBH genes and obtain a detailed classification of the ALKBH gene family in soybean, a neighbor-joining phylogenetic tree was constructed using MEGA 11 software.The tree was based on the multiple alignment of thirteen-six protein sequences from Arabidopsis thaliana (14 members) and G. max (22 members) (Supplementary Table S1), the GmALKBH genes were named according to their order on the chromosome in reference to the Arabidopsis ALKBH gene family.As shown in Figure 1, all of the ALKBH genes were divided into seven classes: ALKBH1, ALKBH2, ALKBH6, ALKBH7, ALKBH8, ALKBH9, and ALKBH10.Among them, ALKBH10 subfamily had the largest number of genes, with eight GmALKBH genes and three AtALKBH genes.The four GmALKBH10B genes could be candidates for m 6 A RNA demethylases due to their close evolutionary relationship with AtALKBH10B (Figure 1).The ALKBH1 subfamily has five GmALKBH genes and four AtALKBH genes.The GmALKBH1 proteins were predicted to be widely localized in the mitochondria, chloroplasts, and nucleus, suggesting that these proteins may have different functions.The remaining classes had a smaller number of genes, including ALKBH2, ALKBH6, ALKBH7, ALKBH8, and ALKBH9.

Genome-Wide Identification of the GmALKBH Genes in Soybean
To extensively identify the ALKBH proteins in soybean (Glycine max (L.) Merr.), a BLASTp analysis in the Phytozome V13 database was conducted using the amino acid sequences of the fourteen known Arabidopsis ALKBH family proteins [7,19].As a result, twenty-four homologous proteins from the genome of G. max (Wm82.a4.v1) were obtained.Among them, twenty-two candidates (Table 1) were identified as GmALKBH family members employing the HMM module (Hidden Markov Model) of the AtALKBH protein sequences from Arabidopsis [20,21].To further investigate the evolutionary relationship between GmALKBH genes and obtain a detailed classification of the ALKBH gene family in soybean, a neighbor-joining phylogenetic tree was constructed using MEGA 11 software.The tree was based on the multiple alignment of thirteen-six protein sequences from Arabidopsis thaliana (14 members) and G. max (22 members) (Supplementary Table S1), the GmALKBH genes were named according to their order on the chromosome in reference to the Arabidopsis ALKBH gene family.As shown in Figure 1, all of the ALKBH genes were divided into seven classes: ALKBH1, ALKBH2, ALKBH6, ALKBH7, ALKBH8, ALKBH9, and ALKBH10.Among them, ALKBH10 subfamily had the largest number of genes, with eight GmALKBH genes and three AtALKBH genes.The four GmALKBH10B genes could be candidates for m 6 A RNA demethylases due to their close evolutionary relationship with AtALKBH10B (Figure 1).The ALKBH1 subfamily has five GmALKBH genes and four AtALKBH genes.The GmALKBH1 proteins were predicted to be widely localized in the mitochondria, chloroplasts, and nucleus, suggesting that these proteins may have different functions.The remaining classes had a smaller number of genes, including ALKBH2, ALKBH6, ALKBH7, ALKBH8, and ALKBH9.These GmALKBH proteins contained 123 to 683 amino acids, and their molecular weights varied from 14.32 to 73.46 kDa.The isoelectric points ranged from 5.71 to 9.86.The subcellular localization of these twenty-two GmALKBH proteins was analyzed using WoLF PSORT prediction.Among these proteins, nineteen GmALKBH proteins were located in the nucleus, one (GmALKBH1A1) in the chloroplast, one (GmALKBH1D) in the mitochondrion, and one (GmALKBH2B) in the extracellular region.Taken together, these results suggested that different GmALKBH proteins may have different biological functions.

Chromosomal Localization and Duplication Analysis of Soybean ALKBH Family Genes
Next, we investigated the chromosomal distribution of the twenty-two GmALKBH genes across the soybean genome and the associated gene duplication events.As shown in Figure 2, all of the GmALKBH genes were randomly distributed on 16 of the 20 soybean chromosomes.Chromosome 9 has three ALKBH family members, chromosomes 8, 14, 19, and 20 each possess two, whereas chromosomes 1, 2, 3, 5, 7, 10, 11, 15, 16, 17, and 18 each contain only one gene.Intriguingly, eight of twenty-two GmALKBH family genes, including GmALKBH1A1, GmALKBH1A2, GmALKBH1B, GmALKBH2A, GmALKBH6B, GmALKBH7, GmALKBH10B3, and GmALKBH10C4, were exclusively distributed on the telomeric regions of the chromosome with low gene density.These results suggested that some GmALKBH genes may have contributed to the expansion and evolution of this gene family.
Various types of gene replication events, including whole genome duplication (WGD) and transposable duplication (TRD), widely occur in plant genomes, leading to the expansion of gene families [22].Collinearity analysis of the GmALKBH family genes revealed that ten gene pairs (GmALKBH1A1/GmALKBH1A2, GmALKBH2A/GmALKBH2B, GmALKBH6A/GmALKBH6B, GmALKBH9B/GmALKBH9C, GmALKBH10B1/GmALKBH10B2, GmALKBH10B1/GmALKBH10B3, GmALKBH10B1/GmALKBH10B4, GmALKBH10C1/ GmALKBH10C2, GmALKBH10C1/GmALKBH10C3, and GmALKBH10C1/GmALKBH10C4) arose from gene duplication events in soybean (Figure 2).Furthermore, a duplication event analysis using a publicly available database [23] suggested that many ALKBH family genes in soybean were the result of whole genome duplication, while others originated from transposable duplication (Table 2).Notably, no tandem duplication events occurred within the GmALKBH gene family.These results indicate that whole genome duplication events might be the reason for the expansion of the GmALKBH gene family.Various types of gene replication events, including whole genome duplication (WGD) and transposable duplication (TRD), widely occur in plant genomes, leading to the expansion of gene families [22].Collinearity analysis of the GmALKBH family genes revealed that ten gene pairs (GmALKBH1A1/GmALKBH1A2, GmALKBH2A/GmALKBH2B, GmALKBH6A/GmALKBH6B, GmALKBH9B/GmALKBH9C, GmALKBH10B1/GmALKBH10B2, GmALKBH10B1/GmALKBH10B3, GmALKBH10B1/GmALKBH10B4, GmALKBH10C1/GmALKBH10C2, GmALKBH10C1/GmALKBH10C3, and GmALKBH10C1/GmALKBH10C4) arose from gene duplication events in soybean (Figure 2).Furthermore, a duplication event analysis using a publicly available database [23] suggested that many ALKBH family genes in soybean were the result of whole genome duplication, while others originated from transposable duplication (Table 2).Notably, no tandem duplication events occurred within the GmALKBH gene family.These results indicate that whole genome duplication events might be the reason for the expansion of the GmALKBH gene family.

Gene Structure and Conserved Motifs of the GmALKBH Family of Genes
We constructed a phylogenetic tree employing the neighbor-joining method to reconstruct the evolutionary relationships among the GmALKBH family genes (Figure 3), which was consistent with that of the phylogenetic analysis constructed using proteins from soybean and Arabidopsis (Figure 1).To investigate the genetic structural diversity among the GmALKBH family genes, an exon-intron distribution analysis was performed.Notably, the members from each subfamily possessed similar gene structures.For example, four members (GmALKBH10C1, GmALKBH10C2, GmALKBH10C3, and GmALKBH10C4) of the GmALKBH10C subfamily had the most exons (8 exons), while GmALKBH7 contained only one exon (Figure 3A).Generally, the candidate genes from different subfamilies were complex and diverse in structure, suggesting that the GmALKBH genes retained their original function and might also expand towards evolving new functions.
We also employed a MEME program to identify the conserved motifs present within the GmALKBH family genes.In total, ten different and highly conserved motifs were observed (Figure 3B and Supplementary Table S2).Motif 2, motif 3, motif 5, and motif 6 were unique to the GmALKBH9 and GmALKBH10 subfamilies, suggesting that they may be important for demethylase activity.However, no conserved motif was detected in GmALKBH2B, possibly because this gene has a new function.Most genes, except GmALKBH10B2, possessed conserved domains, including the 2OG-Fe(II)_Oxy, 2OG-Fe(II)_Oxy2, 2OG-Fe(II)_Oxy superfamily, or AlkB superfamily.The homologous proteins of each subfamily share identical conserved motifs, suggesting that they may be functionally redundant.

Cis-Element Analysis of the GmALKBH Family of Genes
To better understand the transcriptional regulatory activities of GmALKBH genes, we predicted the cis-elements within 2000 bp promoter regions of GmALKBH genes by employing the PlantCARE service website analysis (PlantCARE, a database of plant promoters and their cis-acting regulatory elements (ugent.be)).In total, cis-elements involved in 23 functional categories were identified in GmALKBH genes (Figure 4A), which could be classified into five groups: light responsive, hormone responsive, environmental stress related, developmental response, and other elements (Figure 4B, right panel).Interestingly, light-responsive elements, which accounted for 50.9% of the total cis-elements (Figure 4), were identified in all of the promoters of the GmALKBH family genes (Figure 4A).Among the hormone-responsive elements, those involved in the abscisic acid (ABA), methyl jasmonate (MeJA), auxin, and gibberellin response were highly abundant.The most frequent environmental stress-related element was anaerobic induction.In addition, cis-elements related to the developmental response and others were also identified (Figure 4).The high abundance of light-, MeJA-, auxin-, and gibberellin-responsive elements identified in the promoters of the GmALKBH family genes suggested that the expression levels of these genes were likely influenced by light and several phytohormones, which in turn may affect the development and environmental stimulus responses of soybean.
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Cis-Element Analysis of the GmALKBH Family of Genes
To better understand the transcriptional regulatory activities of GmALKBH genes, we predicted the cis-elements within 2000 bp promoter regions of GmALKBH genes by employing the PlantCARE service website analysis (PlantCARE, a database of plant promoters and their cis-acting regulatory elements (ugent.be)).In total, cis-elements involved in 23 functional categories were identified in GmALKBH genes (Figure 4A), which could be classified into five groups: light responsive, hormone responsive, environmental stress quent environmental stress-related element was anaerobic induction.In addition, cis-elements related to the developmental response and others were also identified (Figure 4).The high abundance of light-, MeJA-, auxin-, and gibberellin-responsive elements identified in the promoters of the GmALKBH family genes suggested that the expression levels of these genes were likely influenced by light and several phytohormones, which in turn may affect the development and environmental stimulus responses of soybean.In Arabidopsis, AtALKBH10B efficiently demethylates m 6 A on RNA [7].To test whether GmALKBH10B1, GmALKBH10B2, GmALKBH10B3, and GmALKBH10B4, orthologues of AtALKBH10B in soybean, are RNA m 6 A demethylases, we independently overexpressed these four GmALKBH10B genes recombinantly in soybean plants (Figure 5A,B), employing a well-established hair roots expression system [24][25][26][27], and detected the m 6 A level on mRNA from root tissue via liquid chromatography-tandem mass spectrometry (LC-MS/MS).Remarkably, the results revealed a significant decrease in m 6 A abundance upon overexpression of the four GmALKBH10Bs compared with that in the control (Figure 5C).These experimental results strongly demonstrate that all four GmALKBH10B proteins possess demethylase activities, showing their ability to remove mRNA m 6 A modifications (Figure 5).

GmALKBH10Bs Are RNA N6-Methyladenosine Demethylases in Soybean
In Arabidopsis, AtALKBH10B efficiently demethylates m 6 A on RNA [7].To test whether GmALKBH10B1, GmALKBH10B2, GmALKBH10B3, and GmALKBH10B4, orthologues of AtALKBH10B in soybean, are RNA m 6 A demethylases, we independently overexpressed these four GmALKBH10B genes recombinantly in soybean plants (Figure 5A,B), employing a well-established hair roots expression system [24][25][26][27], and detected the m 6 A level on mRNA from root tissue via liquid chromatography-tandem mass spectrometry (LC-MS/MS).Remarkably, the results revealed a significant decrease in m 6 A abundance upon overexpression of the four GmALKBH10Bs compared with that in the control (Figure 5C).These experimental results strongly demonstrate that all four GmALKBH10B proteins possess demethylase activities, showing their ability to remove mRNA m 6 A modifications (Figure 5).

Spatiotemporal Expression and Subcellular Localization of GmALKBH10B Subfamily Members
By integrating data from the SoyOmic website (https://ngdc.cncb.ac.cn/soyomics/index, accessed on 5 July 2023), we next investigated the spatial expression patterns of all of the GmALKBH10B genes across various tissues from G.max, including seeds, roots, lateral roots, nodules, stems, leaves, and flowers.The results show distinct expression patterns among these four genes across different tissue types (Supplementary Figure S1).

Spatiotemporal Expression and Subcellular Localization of GmALKBH10B Subfamily Members
By integrating data from the SoyOmic website (https://ngdc.cncb.ac.cn/soyomics/ index, accessed on 5 July 2023), we next investigated the spatial expression patterns of all of the GmALKBH10B genes across various tissues from G.max, including seeds, roots, lateral roots, nodules, stems, leaves, and flowers.The results show distinct expression patterns among these four genes across different tissue types (Supplementary Figure S1).Intriguingly, we observed that GmALKBH10B3 was significantly up-regulated in flowers Plants 2024, 13, 2491 10 of 17 and lateral roots, while GmALKBH10B2 was relatively increased in nodules and stems, demonstrating its potential involvement in different developmental processes in soybean.Furthermore, a protein sequence alignment (Supplementary Figure S2) and motif analysis (Figure 3B) of the GmALKBH10B subfamily revealed high similarity and consistent motif distribution in conserved regions, with no significant differences noted.In contrast, all four genes exhibited low expression levels in seeds, suggesting that GmALKBH10Bs-mediated RNA m 6 A demethylation might not be required for seed development.
In addition, we used a publicly available database (WoLF PSORT, https://wolfpsort.hgc.jp/, accessed on 29 July 2023) to predict the subcellular localization of GmALKBH10B proteins and other members of the GmALKBH10B family.The results show that GmALKBH10B subfamily members, like most other GmALKBH proteins, were all localized in the nucleus (Table 1).To validate this prediction, C-terminal yellow fluorescent protein (YFP) was fused with the full-length amino acid sequences of four GmALKBH10B members and then independently transiently co-expressed with the nuclear marker Histone 2B fused with mCherry (H2B-RFP) in Nicotiana benthamiana leaves.The results reveal that all four proteins predominantly accumulated in the nucleus (Figure 6), which is in line with our prediction.
Plants 2024, 13, x FOR PEER REVIEW 10 of 17 Intriguingly, we observed that GmALKBH10B3 was significantly up-regulated in flowers and lateral roots, while GmALKBH10B2 was relatively increased in nodules and stems, demonstrating its potential involvement in different developmental processes in soybean.Furthermore, a protein sequence alignment (Supplementary Figure S2) and motif analysis (Figure 3B) of the GmALKBH10B subfamily revealed high similarity and consistent motif distribution in conserved regions, with no significant differences noted.In contrast, all four genes exhibited low expression levels in seeds, suggesting that GmALKBH10Bs-mediated RNA m 6 A demethylation might not be required for seed development.
In addition, we used a publicly available database (WoLF PSORT, https://wolfpsort.hgc.jp/,accessed on 29 July 2023) to predict the subcellular localization of GmALKBH10B proteins and other members of the GmALKBH10B family.The results show that GmALKBH10B subfamily members, like most other GmALKBH proteins, were all localized in the nucleus (Table 1).To validate this prediction, C-terminal yellow fluorescent protein (YFP) was fused with the full-length amino acid sequences of four GmALKBH10B members and then independently transiently co-expressed with the nuclear marker Histone 2B fused with mCherry (H2B-RFP) in Nicotiana benthamiana leaves.The results reveal that all four proteins predominantly accumulated in the nucleus (Figure 6), which is in line with our prediction.

Expression Patterns of GmALKBH10B Genes in Response to Abiotic Stress
The results of the cis-element analyses suggest that the expressions of GmALKBH10B subfamily genes were likely regulated by different abiotic stresses.We therefore analyzed the expression levels of GmALKBH10B1, GmALKBH10B2, GmALKBH10B3, and GmALKBH10B4 in the leaves of 2-week-old soybean plants grown under normal conditions or different stress conditions, including heat, cold, drought, salinity, and alkalinity, over 24 h.Generally, abiotic stress treatments significantly altered the expression patterns of four GmALKBH10B genes (Figure 7).However, each gene showed a different response to those stress conditions.For instance, the expression of GmALKBH10B2 and GmALKBH10B3 increased after 6 h of cold treatment, whereas the expression of

Expression Patterns of GmALKBH10B Genes in Response to Abiotic Stress
The results of the cis-element analyses suggest that the expressions of GmALKBH10B subfamily genes were likely regulated by different abiotic stresses.We therefore analyzed the expression levels of GmALKBH10B1, GmALKBH10B2, GmALKBH10B3, and GmALKBH10B4 in the leaves of 2-week-old soybean plants grown under normal conditions or different stress conditions, including heat, cold, drought, salinity, and alkalinity, over 24 h.Generally, abiotic stress treatments significantly altered the expression patterns of four GmALKBH10B genes (Figure 7).However, each gene showed a different response to those stress conditions.For instance, the expression of GmALKBH10B2 and GmALKBH10B3 increased after 6 h of cold treatment, whereas the expression of GmALKBH10B1 and GmALKBH10B4 increased after only 24 h and 2 h, respectively, compared with that of the control.In addition, the expression levels of GmALKBH10B1, GmALKBH10B2, and GmALKBH10B3 remained unchanged until 24 h after alkalinity treatment.In the drought group, the expression of both GmALKBH10B1 and GmALKBH10B3 significantly increased after 2 h, whereas GmALKBH10B4 expression decreased after 24 h compared with that in the control group (Figure 7).Taken together, these results revealed that GmALKBH10Bs-mediated RNA m6A demethylation likely participates in the abiotic stress response in soybean.
Plants 2024, 13, x FOR PEER REVIEW 11 of 17 GmALKBH10B1 and GmALKBH10B4 increased after only 24 h and 2 h, respectively, compared with that of the control.In addition, the expression levels of GmALKBH10B1, GmALKBH10B2, and GmALKBH10B3 remained unchanged until 24 h after alkalinity treatment.In the drought treatment group, the expression of both GmALKBH10B1 and GmALKBH10B3 significantly increased after 2 h, whereas GmALKBH10B4 expression decreased after 24 h compared with that in the control group (Figure 7).Taken together, these results revealed that GmALKBH10Bs-mediated RNA m6A demethylation likely participates in the abiotic stress response in soybean.

Discussion
RNA demethylation mediated by ALKBH family proteins plays a crucial role in the regulation of plant growth, development, and abiotic stress responses [19,[28][29][30].However, the molecular mechanism of the ALKBH genes, especially in legume plants, has remained unclear.In this study, we identified 22 GmALKBH proteins in soybean via the alignment of homologous amino acid sequences of ALKBH in (Figure 1).These homologous ALKBHs were assigned to seven subfamilies, namely ALKBH1, ALKBH2, ALKBH6, ALKBH7, ALKBH8, ALKBH9, and ALKBH10, which was in line with previous analyses in tomato and Populus [28,31].Protein function is frequently conserved between paralogs [32].Similar to the function of AtALKBH10B in Arabidopsis [7], we demonstrated the m 6 A demethylase activities of the four GmALKBH10B proteins by an in vivo assay employing a transient expression system in the hair roots of soybean plants (Figure 5).Simultaneously, we performed a subcellular localization analysis of four GmALKBH10B proteins, which indicated that they were all localized in the nucleus (Figure 6), in line with our prediction (Table 1).
Unlike the known AtALKBH family genes, more GmALKBHs were identified from G. max.Gene duplications are considered to be one of the main driving forces of genetic evolution in the paleopolyploid soybean genome [33].Different gene replication events, such as tandem, fragment duplication, and transposition events, frequently occur in the soybean genome, resulting in the expansion of gene families [22].Ten gene pairs from the GmALKBH family were involved in gene duplication events (Figure 2).Many whole genome duplications and few transposable duplication events, but not tandem duplication events, contributed to the expansion of the GmALKBH gene family (Table 2).
In addition, the gene structures of all GmALKBH family genes were analyzed, with similar structures and conserved motifs present among the members of each subfamily, suggesting a shared evolutionary origin and potential functional similarity among the subfamily members.However, variations in gene structure could still be observed within the same subfamily, which might arise from spontaneous mutations or transposed duplicationinduced genetic information loss occurring in the soybean genome (Table 2).Such changes in gene structure encompass alterations or losses of genetic content within intron-exon regions and potentially influence gene function (Figure 3).
In plants, RNA methylation (m 6 A) is involved in the response to abiotic stresses [17,19,[34][35][36].However, whether GmALKBH10Bs, an RNA N6-methyladenosine demethylase-encoding gene, respond to abiotic stress conditions remains unknown.In our study, cis-elements on 2000 bp promoter sequences of all GmALKBH family genes were analyzed, and the results suggested that these genes might respond to light, phytohormones, and environmental stresses (Figure 4).In particular, ABA response elements (ABREs) were highly enriched in the promoter regions of many GmALKBH family genes, including GmALKBH10B1, GmALKBH10B2, and GmALKBH10B4.These data suggest that several ALKBH genes might be the targets of ABA signaling regulation in soybean.ABA signaling is well known to play an important role in the response to various stress conditions, such as drought, salinity, and cold stresses [33].Hence, GmALKBH genes might be involved in ABA-mediated stress responses.This hypothesis has been confirmed in Arabidopsis and tomato [30,37,38].In Arabidopsis, alkbh10b mutants are sensitive to ABA, osmotic, and salt stress during seed germination [30].Similarly, the knock-out mutant of SlALKBH10B increased sensitivity to ABA treatment and up-regulation of gene expression related to ABA synthesis and response [38].In addition, our qPCR analysis revealed that GmALKB10B genes were likely involved in cold, drought, and alkalinity stimuli.
In conclusion, our study identifies twenty-two GmALKBH family genes in soybean and demonstrates the enzymatic activities of four GmALKBH10Bs, providing new insights into their evolutionary process, genetic diversity, and potential functions in response to abiotic stress.These results serve as the foundation for future research on elucidating the molecular mechanisms underlying GmALKBH-mediated RNA demethylation in response to abiotic stress.

Plant Materials and Abiotic Treatment
Soybean (Glycine max, Williams 82) seeds were cultivated on full-nutrient soil in the greenhouse under the following conditions: 16/8 h day/night cycle, 25 • C/24 • C day/night temperature, 70% humidity, and 100 µmol m −2 −1 light intensity.For this study, 13-dayold plants were used as research materials, and different abiotic treatments were applied.For the cold and heat treatments, soybean seedlings were exposed to temperatures of 8 • C or 42 • C for a duration of 24 h.To induce drought stress, the seedlings were transferred to a Hoagland liquid culture supplemented with 20% polyethylene glycol (PEG, Sangon Biotech, Shanghai, China) and incubated for one day.For salinity and alkalinity stresses, the plants were grown in Hoagland liquid culture containing 150 mM NaCl (Sangon Biotech, Shanghai, China) or 100 mM NaHCO 3 (Sangon Biotech, Shanghai, China), respectively, also for one day.Leaves from single plants were collected at 0 h, 2 h, 6 h, 12 h, and 24 h after treatment.All samples were immediately frozen in liquid nitrogen after harvesting, and were stored at −80 • C until use.

Identification of GmALKBH Gene Family Members and Construction of the Phylogenetic Tree of the ALKBH Family
A BLASTp analysis and a Hidden Markov Model (HMM) based on published AtALKBH protein sequences from Arabidopsis were constructed to search for soybean genomes (Glycine max Wm82.a4.v1) in Phytozome v13 database (https://phytozome-next.jgi.doe.gov/, accessed on 24 June 2023).After removing repeated sequences, a total of 22 putative candidates, including gene IDs and full-length amino acid sequences, were obtained.The CD-Search and InterPro programs were used to detect and confirm the presence of conserved domains in each identified sequence.The molecular weight (MW) and isoelectric point (pI) were predicted using the ExPASy tool.
The protein sequences of the ALKBH gene family from A. thaliana and G. max were used to construct a phylogenetic tree using MEGA11 [39] with the neighbor-joining (NJ) method and 1000 bootstrap replications.Thereafter, the phylogenetic tree was visualized using ChiPlot (https://www.chiplot.online/,accessed on 15 August 2023) [40].

Chromosomal Location and Duplication Analysis of the GmALKBH Gene Family
The physical position of each GmALKBH gene in the Glycine max Wm82.a4.v1 genome annotation was used to determine its chromosomal localization.The GFF data, including length information for all soybean chromosomes, from Phytozome were used to extract the positional information and gene density profiles of the GmALKBH genes on the chromosomes.The three datasets were then uploaded to TBtools for the analysis of GmALKBH genes' chromosomal positions.To investigate the collinearity relationships among GmALKBH genes, the One Step MCScanX-Super Fast program integrated into TBtools was employed.All GmALKBH genes were categorized based on whole genome duplication (WGD) and transposable duplication (TRD) [41].

Gene Structure, Conserved Domain, Motif Analysis, and Cis-Element Analysis of GmALKBH Genes in Soybean
Gene structure analysis was performed based on GFF data extracted from Phytozome (https://phytozome-next.jgi.doe.gov/,accessed on 15 August 2023) to determine UTRs, exons, and introns.Conserved motifs of GmALKBH proteins were identified using the online service MEME (https:/meme-suite.org/meme/,accessed on 17 August 2023) with a maximum of ten motifs.The conserved domains of the GmALKBH protein sequences were identified using the Batch CD-Search program.For the cis-element analysis, the promoter region for each GmALKBH gene was defined as the 2000 base pairs upstream of the genomic sequence from the translation initiation site.The PlantCARE software (https://bioinformatics.psb.ugent.be/webtools/plantcare/html/,accessed on 17 August 2022) was used for prediction.Four types of analysis were visualized using the TBtools software (v2.121).

Soybean Hairy Root Transformation
A modified soybean hairy-root transformation method was utilized based on a previous study [24][25][26][27] to generate transgenic soybean lines.Transformed Agrobacterium rhizogenes strain K599 cells were streaked onto the surface of a TY medium containing the appropriate antibiotic and incubated at 28 • C until the appropriate bacterial growth state and cell density were reached.The soybean plants, which were grown to the state of spreading true leaves, were used for the transformation experiments.The plants were cut diagonally at 45 • at 0.5-1 cm below the soybean cotyledons, and the bacterial chunks were evenly spread on the wounds using an aseptic loop.After a day of recovery in the dark, the plants were placed on dampened vermiculite and cultivated under normal growth conditions.After 15 days, the hairy roots were analyzed using an MVX10 microscope (Olympus Corporation, Tokyo, Japan), and the positive hairy roots displaying GFP fluorescence signals were collected and frozen at −80 • C for subsequent experiments.

Subcellular Localization and Tissue-Specific Expression of GmALKBH10B Genes
GmALKBH10B-eYFP was transiently co-expressed with the nuclear marker protein H2B-RFP (RFP fused to histone 2B) [47] in Nicotiana benthamiana leaves for 5 days.The samples were analyzed using a ZEISS LSM 980 instrument with an Airyscan2 microscope equipped with an HC PLAPO CS2 40 × 1.0 water immersion objective (ZEISS Microsystems) according to a previous description [48].The data on the tissue-specific expression of GmALKBH10B, including flowers, lateral roots, leaves, nodules, roots, seeds, and stems, were downloaded from the transcriptome module of the Soyomics database (https://ngdc.cncb.ac.cn/soyomics/index, accessed on 15 August 2022) and visualized using Chiplot (https://www.chiplot.online/,accessed on 15 August 2022).

Figure 1 .
Figure 1.Phylogenetic analysis of ALKBH family proteins in Arabidopsis thaliana and Glycine max.An unrooted phylogenetic tree was generated via the neighbor-joining method via MEGA11 software; 1000 bootstraps.

Figure 1 .
Figure 1.Phylogenetic analysis of ALKBH family proteins in Arabidopsis thaliana and Glycine max.An unrooted phylogenetic tree was generated via the neighbor-joining method via MEGA11 software; 1000 bootstraps.

Figure 3 .
Figure 3. Gene structure, conserved domain, and motif analysis of the GmALKBH family genes.(A) Phylogenetic analysis and exon-intron structures of the GmALKBH family genes.The green boxes, yellow boxes, and gray lines represent untranslated regions (UTRs), coding sequences (CDSs), and introns, respectively.(B) Phylogenetic analysis and the conserved domain and motif distributions of the GmALKBH family genes.

Figure 3 .
Figure 3. Gene structure, conserved domain, and motif analysis of the GmALKBH family genes.(A) Phylogenetic analysis and exon-intron structures of the GmALKBH family genes.The green boxes, yellow boxes, and gray lines represent untranslated regions (UTRs), coding sequences (CDSs), and introns, respectively.(B) Phylogenetic analysis and the conserved domain and motif distributions of the GmALKBH family genes.

Figure 4 .
Figure 4. Prediction of cis-elements in the GmALKBH family gene promoter.(A) Cis-element distribution of the GmALKBH family gene promoter.Different cis-elements are represented by different colors.(B) The classification of the cis-elements and the proportions of different types of cis-elements.The 519 cis-elements were divided into five groups, including 269 light-responsive elements, 136 hormone-responsive elements, 62 environmental stress-related elements, 35 developmental responsive elements, and 17 other elements.

Figure 4 . 17 2. 5 .
Figure 4. Prediction of cis-elements in the GmALKBH family gene promoter.(A) Cis-element distribution of the GmALKBH family gene promoter.Different cis-elements are represented by different colors.(B) The classification of the cis-elements and the proportions of different types of cis-elements.The 519 cis-elements were divided into five groups, including 269 light-responsive elements, 136 hormoneresponsive elements, 62 environmental stress-related elements, 35 developmental responsive elements, and 17 other elements.

Figure 6 .
Figure 6.Subcellular localization of GmALKBH10B proteins in Nicotiana benthamiana leaves.Green represents YFP, red represents RFP, and yellow represents the overlap of green and red.Bar, 100 μM.BF, brightfield.

Figure 6 .
Figure 6.Subcellular localization of GmALKBH10B proteins in Nicotiana benthamiana leaves.Green represents YFP, red represents RFP, and yellow represents the overlap of green and red.Bar, 100 µM.BF, brightfield.

Table 1 .
Protein characteristics of the predicted ALKBH candidate genes in Glycine max.

Table 2 .
Gene duplication identified in the GmALKBH gene family.