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Article

Genome-Wide Identification and Expression Analysis of the AlkB Homolog Gene Family in Tamarix chinensis

by
Jingjing Zhang
1,2,3,
Wenhui Guo
1,2,3,
Huijuan Yin
1,2,3,
Kongshu Ji
1,2,3 and
Qiong Yu
1,2,3,4,*
1
State Key Laboratory of Tree Genetics and Breeding, Nanjing Forestry University, Nanjing 210037, China
2
Key Open Laboratory of Forest Genetics and Gene Engineering of National Forestry & Grassland, Nanjing Forestry University, Nanjing 210037, China
3
Co-Innovation Center for Sustainable Forestry in Southern China, Nanjing Forestry University, Nanjing 210037, China
4
Beijing National Laboratory for Molecular Sciences, Beijing 100190, China
*
Author to whom correspondence should be addressed.
Forests 2025, 16(3), 470; https://doi.org/10.3390/f16030470
Submission received: 3 February 2025 / Revised: 28 February 2025 / Accepted: 6 March 2025 / Published: 7 March 2025
(This article belongs to the Section Genetics and Molecular Biology)
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Abstract

:
Tamarix chinensis (T. chinensis), an esteemed salt-tolerant plant, holds significant importance in elucidating mechanisms of plant stress adaptation. The ALKBH genes family, which is involved in RNA N6-methyladenosine (m6A) demethylation, plays a crucial role in plant growth, development, and stress responses. This study performed a genome-wide identification and analysis of the ALKBH genes family in T. chinensis using bioinformatics methodologies. A total of eight ALKBH genes were identified and named TcALKBH1 to TcALKBH8 based on their chromosomal positions. Phylogenetic analysis divided the TcALKBH genes family into different subgroups, revealing that, in comparison to Arabidopsis and other plants, T. chinensis lacks members of the ALKBH6 and ALKBH10 families. Further analysis of gene structure, conserved domain, and motif analysis elucidated the basic features of the TcALKBH gene family. Gene duplication analysis identified TcALKBH3 and TcALKBH7 as homologous gene pairs, and collinearity analysis indicated a closer relationship between T. chinensis and Populus compared to Arabidopsis. In addition, gene expression analysis revealed tissue-specific expression patterns of the TcALKBH genes, with significant upregulation observed under abiotic stress conditions such as ABA, NaCl, and NaHCO3. It is noteworthy that the expression of TcALKBH4 increased nearly 30-fold after 6 h of ABA stress, suggesting that TcALKBH4 may play a key regulatory role in the ABA response. These results indicate that the TcALKBH genes might be crucial for stress responses in T. chinensis. This research offers a theoretical foundation for a deeper exploration of the roles and molecular mechanisms of the TcALKBH genes family in stress adaptation. It also presents valuable candidate genes for enhancing stress resistance in plants through breeding programs.

1. Introduction

N6-methyladenosine (m6A), the most prevalent post-transcriptional chemical modification in eukaryotic RNA molecules, is widely distributed in mRNAs, lncRNAs, circRNAs, and other non-coding RNAs [1]. It regulates RNA metabolism and functions through dynamic and reversible methylation–demethylation processes. This modification is precisely orchestrated by “Writers” (methyltransferases), “Erasers” (demethylases), and “Readers” m6A-binding proteins), which collectively influence RNA splicing, stability, trafficking, and translational efficiency [1,2]. Recent research has shown that m6A plays pivotal roles in plant growth, stress adaptation, and epigenetic regulation. ALKBH-mediated demethylation dynamically balances RNA homeostasis during stress signaling [3,4,5,6]. As a core mechanism of RNA epigenetic regulation, m6A research offers novel insights into deciphering the molecular networks underlying plant adaptation to complex environments.
The AlkB homologs (ALKBH) family consists of Fe(II) and α-ketoglutarate-dependent dioxygenases, involved in the oxidative demethylation of DNA and RNA [7,8]. These enzymes were initially discovered in Escherichia coli, where they are capable of removing methyl groups generated by alkylation damage, thereby repairing DNA lesions caused by alkylation [9,10]. With the discovery of ALKBH proteins in various organisms, researchers have gradually recognized their crucial role in maintaining genomic stability and responding to environmental stress [11]. These proteins not only repair DNA damage caused by alkylating agents but also regulate RNA stability and function, playing a regulatory role in plant growth, development, and environmental adaptation [3,4,5,6,12,13]. Gaining a more comprehensive insight into the molecular mechanisms of ALKBH homologs will offer novel approaches for areas like the development of plant stress resistance.
Although Escherichia coli contains only one AlkB gene, multiple AlkB homologs are present in higher organisms, including animals and plants [7]. Each homolog exhibits distinct biological activities in repairing specific types of damage. These homologs not only participate in DNA repair but also play important physiological roles by regulating RNA stability and function. For example, in plants, ALKBH proteins have been identified as enzymes that function to remove m6A methylation and are involved in the regulation of RNA modifications that affect plant development and response to environmental stress [3]. In Arabidopsis thaliana, ALKBH10B mediates the m6A demethylation of the flowering activator FT and its upregulating factors SPL3 and SPL9, affecting the stability of their mRNA and thereby regulating the floral transition [3]. During Arabidopsis thaliana seed germination, ALKBH10B negatively regulates ABA response genes to control seed germination [14]. Furthermore, research demonstrates a molecular connection between ALKBH10B-directed m6A demethylation and the regulation of mRNA stability, which plays a role in drought tolerance [15]. Overexpression of ALKBH10B enhances drought tolerance in plants. ALKBH9B in Arabidopsis thaliana can remove m6A from Medicago sativa mosaic virus RNA and plays a role during Arabidopsis thaliana infection with the virus [16]. The deletion of ALKBH9B results in increased sensitivity to ABA treatment during seed germination and the early stages of seedling development [6].
T. chinensis is a plant with strong salt and alkali tolerance, widely distributed in extreme environments such as saline–alkali soils, deserts, and wetlands [17,18]. It demonstrates remarkable adaptability in saline–alkali soils, effectively improving the salinity of the soil and promoting ecological restoration. T. chinensis maintains good growth in saline–alkali environments through multiple mechanisms such as osmotic regulation by its root system, ion balance adjustment, and enhanced antioxidant capacity, thus providing important support for the ecological restoration and agricultural productivity of saline–alkali lands. Given the potential of T. chinensis in saline–alkali land improvement, studying its molecular mechanisms under salt–alkali stress is of great significance. The function of the TcALKBH genes family in T. chinensis under salt–alkali stress remains to be fully elucidated. However, current studies indicate that members of the ALKBH genes family are crucial for plants in coping with salt–alkali stress. For example, Arabidopsis thaliana ALKBH6 knockdown mutants exhibit faster seed germination under low temperature, salt, or ABA treatments compared to wild-type seeds. ALKBH8B overexpression transgenic lines show enhanced salt tolerance under salt stress [4], with better seedling growth and survival rates compared to wild-type; ALKBH9C is critical in regulating the stability of stress-responsive transcripts [5], which is essential for seed germination and seedling growth in Arabidopsis thaliana under salt or ABA stress. Potato SlALKBH10B mutants demonstrate enhanced drought and salt stress tolerance [19], with increased water retention, photosynthetic product accumulation, and proline accumulation, alongside reduced reactive oxygen species and cell damage. Overexpression of PagALKBH9B and PagALKBH10B in poplar leads to enhanced activities of superoxide dismutase (SOD), peroxidase (POD), and catalase (CAT). This results in decreased accumulation of hydrogen peroxide (H2O2) and reduced oxidative damage, consequently improving the salt tolerance of the transgenic lines [20].
By identifying the TcALKBH genes family in T. chinensis, the specific mechanisms of these genes in salt–alkali tolerance can be uncovered, providing important insights into T. chinensis adaptation strategies in saline–alkali environments. Furthermore, an in-depth study of the TcALKBH genes family may offer theoretical support for the breeding and improvement of salt–alkali tolerant plants, helping to enhance plant survival under harsh environmental conditions and providing new solutions for ecological restoration and sustainable agricultural development.

2. Materials and Methods

2.1. Identificaiton of ALKBH Genes in the T. chinensis Genome

To identify and characterize the TcALKBH genes family in T. chinensis, this study first downloaded the genome and protein sequences of T. chinensis from NCBI (https://ftp.ncbi.nlm.nih.gov/genomes/all/GCA/030/549/775/GCA_030549775.1_ASM3054977v1/, accessed on 5 November 2024) [18]. Known ALKBH protein sequences from Arabidopsis thaliana were used for BLASTp searches [21]. In addition, the Pfam database (https://www.ebi.ac.uk/interpro/entry/pfam/, accessed on 5 November 2024) was used to download the 2 g-fe (II) oxygenase superfamily (PF13532) HMM file, and the identification was made with the HMMER 3.3.2 software [22,23]. The results from both BLASTp and HMMER were then combined, and redundant sequences were removed. Use the Pfam and CDD tools (https://www.ncbi.nlm.nih.gov/cdd, accessed on 6 November 2024) to verify the candidate proteins of the key domain structure. The TcALKBH genes are named sequentially as TcALKBH1-8 based on their positions on the chromosome. Protein characteristics were analyzed utilizing the ProtParam (https://web.expasy.org/protparam/, accessed on 6 November 2024) [24], and the WoLF PSORT (https://wolfpsort.hgc.jp/, accessed on 7 November 2024) was used to predict subcellular localization.

2.2. Chromosomal Mapping and Phylogenetic Analysis of TcALKBH Genes

We extracted chromosomal location information and chromosomal gene density information of TcALKBH genes in T. chinensis using a genome annotation file. The chromosomal location of the TcALKBH genes was subsequently visualized utilizing the TBtools-II v2.154 software [25]. To explore the evolutionary connections within the TcALKBH genes family, we selected ALKBH protein sequences from T. chinensis, Arabidopsis thaliana, Triticum aestivum, Zea mays, Vitis vinifera, and Populus alba × Populus tremula var. glandulosa for multiple sequence alignment using MEGA 11 software [26]. The phylogenetic tree was built using the NJ (Neighbor-Joining) method set to 1000 bootstrap repetitions. The evolutionary tree was then visualized and enhanced using the Evolview v3 online tool (https://www.evolgenius.info/evolview/, accessed on 10 November 2024) [27].

2.3. Gene Structure, Amino Acid Sequence and Gene Collinearity Analysis

TBtools-II was used to extract TcALKBH gene structure information from genome annotation files. The CDD tool was used to predict conserved domains, and the MEME tool (https://meme-suite.org, accessed on 11 November 2024) was used to identify motifs [28]. The gene structure, conserved domains, and motif distributions were then visualized using TBtools-II. Gene duplication events in T. chinensis were investigated utilizing the MCScanX tool within the TBtools-II package, and inter-species and intra-species analysis of the YTH family members in T. chinensis, Arabidopsis thaliana, and Populus trichocarpa was conducted. The fragment duplication relationships were visualized using the Advanced Circos function.

2.4. Cis-Acting Elements Analysis of the TcALKBH Genes Promoter

The 2000 bp upstream sequence of the TcALCBH genes promoter was extracted, and the cis-acting of the TcALCBH genes promoter region was predicted by PlantCARE [29]. After manually classifying and filtering out nonspecific elements, the specific positions and numbers of the selected cis-acting elements in the promoter region were visualized using Tbtools-II.

2.5. Plant Materials and Stress Treatment

One-year-old T. chinensis cuttings were provided by the CAS Engineering Laboratory for Efficient Utilization of Saline Resources. These plants were cultivated at the State Key Laboratory of Tree Genetics and Breeding, Nanjing Forestry University. The plants were cultivated in containers filled with 1.3 L of fertilized soil, which consisted of a mixture of peat, perlite, and vermiculite in a ratio of 3:1:1 (by volume). Watering was performed on a weekly basis. The environmental parameters for growth were maintained at a temperature of 25 °C, featuring a photoperiod of 16 h of light and 8 h of darkness, alongside a relative humidity level of 70%. To investigate the tissue-specific expression profile of TcALKBH genes, fresh samples from roots, stems, and leaves were gathered for analysis. For stress response evaluation, different treatments were applied [30]. Each plant was sprayed with 50 mL of 100 µM ABA solution and irrigated with 1 L of 300 mM NaCl solution and 1 L of 300 mM NaHCO3 solution for the respective treatments. Leaf samples were collected at five time points after treatment: 0, 6, 12, 24, and 48 h, with the 0 h samples serving as the control. All treatments were conducted with three biological replicates. Samples collected were promptly frozen in liquid nitrogen and stored at −80 °C for later RNA extraction.

2.6. qRT-PCR Was Used to Analyze the Expression of TcALKBH Genes

To isolate total RNA, we followed the manufacturer’s protocol using the Plant Total RNA Extraction Kit (Vazyme, Nanjing, China). The RNA that was extracted was evaluated for its concentration and purity utilizing a NanoDrop OneC spectrophotometer (Thermo Fisher Scientific, Waltham, MA, USA). To verify the quality of the RNA, we ensured that the A260/A280 ratio fell within the range of 1.8 to 2.0. Additionally, RNA integrity was further confirmed by running a 1% agarose gel electrophoresis, ensuring that the RNA was not degraded and exhibited clear, intact bands. To eliminate genomic DNA contamination, we first used DNA removal enzymes to remove potential DNA contamination and then converted RNA into cDNA using a first-strand cDNA synthesis kit (Yeasen, Shanghai, China). The 1st strand cDNA synthesis reaction was conducted under optimal conditions as specified by the kit instructions. Following cDNA synthesis, qRT-PCR was performed using SYBR Green Master Mix (Yeasen, Shanghai, China) with specific primers designed for the target gene and reference gene. During the melt curve stage, the instrument operates using the default parameters specified by the manufacturer: a temperature of 95 °C for 15 s, 60 °C for 1 h, and 95 °C for 15 s. Potential contamination or nonspecific amplification is detected using a no-template control and a no-reverse-transcription control to ensure the accuracy of the results. Melt curve analysis is performed to confirm the specificity of the PCR products and to prevent any contamination (Figure S1). TIF (translation initiation factor) and α-tubulin genes are used as normalized reference genes [18]. A primer list is provided in the Supplementary Materials (Tables S6 and S7). Three technical replicates were performed for each sample to ensure reliable results. The relative abundance of transcripts was calculated using the 2−ΔΔCt method to assess the expression changes in target genes under different treatments [31]. GraphPad Prism 9.5 software was used for mapping and statistical analysis. Statistical significance was assessed using Duncan’s multiple range test, where different letters denote significant differences at the p < 0.05 level. The asterisk signifies a statistically significant difference relative to the control group, as determined by Student’s t-test (* p < 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.0001).

3. Results

3.1. Genome-Wide Identification of TcALKBH Gene in T. chinensis

In this study, a comprehensive analysis of the T. chinensis genome led to the identification of eight TcALKBH genes and named TcALKBH1 to TcALKBH8 based on their chromosomal locations (Figure 1; Table 1 and Table S1). These genes are distributed across multiple chromosomes. Specifically, chromosomes 10 and 11 each contain two TcALKBH genes, while chromosomes 1, 8, 9, and 12 each harbor one TcALKBH gene (Figure 1). The proteins encoded by the T. chinensis TcALKBH genes family range from 165 to 524 amino acids in length, with molecular weights (MW) ranging from 18.67 kDa to 59.26 kDa. The isoelectric points (pI) of these proteins vary between 4.75 and 9.26. Amino acid composition analysis revealed that all TcALKBH proteins have negative Grand Average of Hydropathicity (GRAVY) values (−0.366 to −0.673), suggesting that they are likely hydrophilic. Additionally, the aliphatic index of TcALKBH proteins ranges from 64.49 to 80.56, indicating a certain degree of protein stability. To further predict the potential cellular functions of TcALKBH genes, this study employed online tools to analyze their subcellular localization. The findings indicated that the majority of TcALKBH proteins are situated within the nucleus (TcALKBH1, TcALKBH2, TcALKBH3, TcALKBH4, and TcALKBH8), suggesting a possible role in the dynamic regulation of RNA modifications. In addition, TcALKBH5 and TcALKBH7 were predicted to be localized in the chloroplast, potentially participating in photosynthesis or oxidative stress-related regulatory functions. Meanwhile, TcALKBH6 is mainly distributed in the cytosol, implying a role in mRNA stability maintenance or protein translation processes.

3.2. Phylogenetic Analysis of TcALKBH Genes

To further explore the evolutionary relationships of the TcALKBH genes family, we included a broader set of plant species, providing a comparative context for understanding the diversification and functional specialization of TcALKBH genes. We constructed a phylogenetic tree based on 8 members from T. chinensis, 13 members from Arabidopsis thaliana, 29 members from Triticum aestivum, 10 members from Zea mays, 11 members from Vitis vinifera, and 23 members from Populus alba × Populus tremula var. glandulosa (Table S2). The results showed that the ALKBH family can be divided into seven groups: ALKBH1, 2, 6, 7, 8, 9, and 10 (Figure 2). Among them, the ALKBH genes from T. chinensis are distributed across ALKBH1, with three genes, ALKBH2 with one gene, ALKBH7 with one gene, ALKBH8 with one gene, and ALKBH9 with two genes, corresponding to five categories. However, there are no members of ALKBH6 and ALKBH10 in T. chinensis, which reflects differences in environmental adaptation and evolutionary divergence.

3.3. Gene Structure, Conserved Domain, and Motif Analysis of TcALKBH Genes

To analyze the functional divergence and evolutionary relationships of the TcALKBH genes family, we conducted analyses of gene structure, conserved domains, and motifs. The findings indicated that the TcALKBH genes family members had a variable number of exons, ranging between three and nine, which may be associated with their functional diversity, evolutionary dynamics, and stress response strategies (Figure 3A). Genes that are closely related evolutionarily tend to share similar exon–intron structures. Moreover, genes that group closely in the phylogenetic tree usually share identical conserved domains and exhibit similar motifs. The conserved 2OG-Fe(II) domain in TcALKBH proteins confirms their classification within the ALKBH superfamily and supports their potential demethylase function (Figure 3B). However, it is noteworthy that the extent of conservation of this domain, along with other motifs, differs slightly between subgroups, which could reflect functional divergence within the family. Moreover, each TcALKBH protein contains motif 4 (Table S3 and Figure 3C), which serves as a Fe2+ binding site, a characteristic feature of the ALKBH genes family. Subgroups that share similar structures and motifs likely perform related functions, while those that exhibit differences in exon number, conserved domains, or motifs may have diverged functionally to address different physiological demands or environmental stresses.

3.4. Gene Replication Analysis and Synteny Analysis of the TcALKBH Genes

Whole-genome duplication, segmental duplication, and tandem duplication are major mechanisms driving gene family expansion and genome evolution, providing critical insights into the structural evolution and regulatory networks of species genomes. In this study, through an analysis of duplication events in the TcALKBH genes family of T. chinensis, we found that TcALKBH3 and TcALKBH7 were paralogous gene pairs derived from segmental duplication of chromosomal regions (Figure 4A), indicating that segmental duplication has been a significant evolutionary force driving the expansion of this gene family. To further analyze the evolutionary pressure on homologous genes, we analyzed the ratio between the non-synonymous substitution rate (Ka) and the synonymous substitution rate (Ks) of TcALKBH3 and TcALKBH7. The results showed that the Ka/Ks ratio is 0.2806, which is less than 1, indicating that TcALKBH3 and TcALKBH7 are undergoing purifying selection (Table S4). This suggests that they are evolutionarily conserved and likely play an important, conserved role in the biology of the organism. To explore the evolutionary paths and functional preservation of the TcALKBH genes family across different species more thoroughly, we performed a genome-wide synteny analysis of T. chinensis, Arabidopsis thaliana, and Populus trichocarpa. The results revealed three pairs of orthologous ALKBH genes between T. chinensis and Arabidopsis thaliana, while four orthologous gene pairs were identified between T. chinensis and Populus trichocarpa (Figure 4B). The higher retention rate of orthologous genes between T. chinensis and Populus trichocarpa suggests a closer evolutionary relationship, likely due to their shared evolutionary adaptation within the order Salicales. In contrast, Arabidopsis thaliana, belonging to the order Brassicales, underwent an earlier divergence, leading to a higher loss of orthologous genes.

3.5. Tissue-Specific Expression Pattern of TcALKBH Genes

To elucidate the potential roles of TcALKBH genes in the growth and development of T. chinensis, we analyzed their tissue-specific expression patterns. Firstly, individual TcALKBH genes exhibited significant expression heterogeneity across different organs. TcALKBH1 showed root-specific high expression, while TcALKBH2, TcALKBH3, and TcALKBH7 had the highest expression in leaves, following a hierarchical pattern of leaf > root > stem, suggesting their potential involvement in leaf-related physiological processes (Figure 5A). TcALKBH8 was predominantly expressed in stems, whereas TcALKBH4, TcALKBH5, and TcALKBH6 were broadly expressed across roots, stems, and leaves without significant differences, indicating more generalized roles. Secondly, different TcALKBH genes exhibit preferential expression dominance within the same tissue. TcALKBH5 has the highest expression level in the leaves, while TcALKBH1 and TcALKBH6 are most highly expressed in the stems, although their expression levels in the leaves are the lowest. The lowest expression level in the stems is observed for TcALKBH2, while TcALKBH4 and TcALKBH5 have the highest expression levels in the roots, and the lowest expression level in the roots is observed for TcALKBH6 (Figure 5B). This dual-dimensional cross-tissue and within-tissue expression pattern suggests that TcALKBH genes functionally diverge through spatiotemporal regulatory specificity, potentially coordinating multi-organ adaptation mechanisms related to halophytic growth and development in T. chinensis.

3.6. Analysis of Promoter Cis-Acting Elements of TcALKBH Genes

Functional classification analysis of the cis-acting regulatory elements in the promoters of the TcALKBH genes family reveals that all genes contain light-responsive elements, with TcALKBH7 and TcALKBH8 having the highest numbers, suggesting that light signals may broadly regulate these genes (Table S5 and Figure 6). In terms of hormone responses, TcALKBH8 is enriched with ABA-responsive elements, TcALKBH1, TcALKBH2, and TcALKBH4 are enriched with MeJA-responsive elements, and TcALKBH5 and TcALKBH6 are enriched with SA-responsive elements, indicating that these genes may mediate different stress signaling pathways. The variation in the response elements associated with each gene points to a functional differentiation among the TcALKBH genes in relation to hormone signaling. This specialization could play a key role in enhancing the plant’s ability to adapt to diverse stress conditions. In terms of stress response elements, family members exhibit significant heterogeneity. For instance, TcALKBH4 contains eight cold-responsive elements, which may be associated with cold adaptation, while TcALKBH7 contains seven hypoxia-inducible elements, which may be involved in flood tolerance. Additionally, TcALKBH8 may enhance salt and alkali adaptation through the synergistic regulation of ABA and light signals. Furthermore, plant growth and development-related response elements were also identified, such as meristem expression (with TcALKBH3 containing two and TcALKBH4/6/8 each containing one) and endosperm expression (with TcALKBH6 and TcALKBH7 each containing one). These elements suggest that TcALKBH genes may play regulatory roles in processes such as the maintenance of apical meristem activity, organ differentiation, and seed development. The distribution patterns of these elements are highly correlated with the genes’ tissue-specific expression patterns and stress response characteristics, providing a regulatory network-level framework for understanding the functional diversity of the TcALKBH genes family in stress tolerance.

3.7. Expression Patterns of TcALKBH Genes Under ABA, NaCl, and NaHCO3 Stress

The analysis of cis-acting elements indicates that the promoters of TcALKBH gene family members in T. chinensis are enriched with elements associated with hormone responses, particularly ABA responses and stress responses. Additionally, the analysis also reveals that several members of the TcALKBH genes family contain elements related to stress response T. chinensis, as a typical salt-tolerant plant, can grow and reproduce in saline–alkaline environments, making it an important model for studying the mechanisms of salt–alkali tolerance. Therefore, we further analyzed the expression patterns of TcALKBH genes under ABA, NaCl, and NaHCO3 stress conditions to better understand the functions and regulatory networks of the TcALKBH genes family under different stress conditions, providing key insights into T. chinensis adaptation mechanisms in extreme environments. Under ABA stress conditions, the expression levels of all eight TcALKBH genes were significantly altered. Except for TcALKBH7 and TcALKBH8, the expression of other genes initially increased and then decreased (Figure 7A). Specifically, TcALKBH7 showed a trend of initially decreasing, followed by a significant increase at 48 h, suggesting a specific regulatory role for this gene in ABA stress response. In contrast, TcALKBH8 expression significantly decreased throughout the stress treatment, indicating its potential inhibitory role in ABA response. Notably, TcALKBH4 expression increased nearly 30-fold at 6 h after ABA stress, suggesting that TcALKBH4 may play a key regulatory role in the ABA response. Under NaCl stress, the expression levels of TcALKBH1, TcALKBH3, TcALKBH4, and TcALKBH6 were significantly altered at 24 or 48 h, showing a relatively slow response to salt stress (Figure 7B). However, TcALKBH2, TcALKBH5, TcALKBH7, and TcALKBH8 began to respond actively to salt stress as early as 6 h, indicating that different TcALKBH genes may have temporal differences in their response to salt stress, reflecting potential functional differences in regulating salt stress. Under NaHCO3 stress conditions, TcALKBH1 and TcALKBH2 showed a significant decrease in expression at 48 h, indicating a delayed response (Figure 7C). Although TcALKBH3, TcALKBH5, TcALKBH6, TcALKBH7, and TcALKBH8 showed significant changes in expression, the magnitude of the changes was small, suggesting that their response to NaHCO3 stress may be milder or regulated in different degrees. In contrast, TcALKBH4 expression showed no significant change, possibly indicating its relatively weaker role in NaHCO3 stress.

4. Discussion

The ALKBH genes family encodes a group of Fe(II) and 2-oxoglutarate-dependent dioxygenases, primarily involved in nucleic acid demethylation [7]. Initially identified in Escherichia coli as an enzyme that repairs alkylation damage in DNA, ALKBH family members in eukaryotes have undergone evolutionary diversification to regulate RNA modifications, particularly m6A demethylation. In plants, ALKBH genes play crucial roles in stress responses, growth, and development by dynamically modulating RNA methylation levels, thereby affecting gene expression and environmental adaptability [3,14,15]. Recent studies have demonstrated that plant ALKBH proteins are involved in abiotic stress regulation, including drought, salt, and hormonal responses, suggesting their significant role in plant resilience to harsh environments [3,4,5,19]. In Arabidopsis thaliana, ALKBH10B regulates the flowering pathway, while ALKBH6 and ALKBH9C play crucial roles in the germination of seeds, the growth of seedlings, and their survival [3,5,12]. ALKBH8B plays a role in the salt stress and ABA response mechanisms of Arabidopsis thaliana by modulating the expression of genes associated with salt stress or ABA signaling pathways [4]. The Solanum lycopersicum SlALKBH10B mutant shows enhanced tolerance to drought and salt stress [19], and SlALKBH2 regulates fruit ripening through redox modification [32].
This study conducted a genome-wide identification and multidimensional analysis to reveal the characteristics of the TcALKBH gene family in T. chinensis. As a typical halophytic plant, T. chinensis has evolved robust mechanisms to acclimate to saline–alkali stress, with the TcALKBH genes family playing an essential role in growth, development, and the transmission of stress signals. A total of eight TcALKBH genes were identified in T. chinensis and named TcALKBH1-8 based on their chromosomal locations (Figure 1). Notably, the size of this gene family is significantly smaller than that of model plants such as Arabidopsis thaliana. Phylogenetic analysis revealed that T. chinensis has undergone gene loss events, specifically within the ALKBH6 and ALKBH10 subfamily during evolution (Figure 2), possibly due to redundant gene elimination or functional compensation mechanisms. This observed gene loss suggests that T. chinensis has adopted a unique evolutionary strategy of gene family contraction and functional plasticity to adapt to extreme environments, providing new insights into the adaptive evolution of plant gene families under stress-driven selection, thereby enhancing our understanding of the genetic basis of salt tolerance in halophytes. Gene structure and motif analysis unveiled substantial variations in the exon–intron structure and conserved domains of the TcALKBH genes, indicating their functional diversification and specialization (Figure 3). All TcALKBH proteins contain the conserved 2OG-Fe(II) domain, firmly establishing their classification within the ALKBH family and confirming their potential RNA demethylation function. Furthermore, the presence of Motif 4, functioning as a Fe2+ binding site, across all TcALKBH proteins aligns with observations in soybean, potato, and orange, underlining a fundamental characteristic of the ALKBH genes family. Gene duplication analysis indicated segmental duplication as a pivotal force driving the expansion of the TcALKBH genes family. Specifically, TcALKBH3 and TcALKBH7 form a pair of paralogous genes derived from segmental duplication (Figure 4A). Additionally, synteny analysis showed that, compared to Arabidopsis thaliana, T. chinensis retains more ALKBH orthologous genes with Populus trichocarpa (Figure 4B), suggesting a closer evolutionary relationship between the two, likely due to their shared adaptation to the Salicales environment. These findings offer valuable perspectives on the evolutionary patterns and functional adjustments of the TcALKBH genes family in stress-tolerant plant species. Analysis of promoter cis-acting elements further corroborates the functional diversification of the TcALKBH genes (Figure 6). Each TcALKBH gene contains a rich array of light-responsive elements, particularly TcALKBH7 and TcALKBH8, indicating that these genes are likely to be widely regulated by light signals. This abundance of light-responsive elements is consistent with findings in other species, reinforcing their potential role in photoregulation [33,34,35]. Moreover, the selective enrichment of hormone-responsive elements (such as ABA, MeJA, and SA response elements) in different TcALKBH genes suggests that they may be involved in a complex regulatory network of stress signaling. Notably, TcALKBH4 contains eight cold-responsive elements, hinting at its potential role in regulating gene expression to help plants maintain growth and physiological function under cold stress. Conversely, TcALKBH7 contains seven hypoxia-regulated elements, which may help plants cope with water stress or soil hypoxia, potentially linking its function to the adaptation of T. chinensis to specific environments such as saline–alkaline wetlands, thereby illuminating its role in environmental acclimation strategies.
To investigate the expression profiles of TcALKBH genes and elucidate their mechanisms in responding to stress conditions, we explored the expression levels of TcALKBH genes. Tissue-specific expression analysis revealed the functional differentiation of TcALKBH genes, highlighting their potential organ-specific roles in the stress response and other physiological processes of T. chinensis (Figure 5A). Notably, TcALKBH1 exhibited predominantly high expression in roots, which indicates that the gene might be crucial for processes specific to root development, such as nutrient absorption, ion transport, or adaptation to soil-based stresses, including salinity or drought. In contrast, TcALKBH2, TcALKBH3, and TcALKBH7 demonstrated peak expression in leaves, implying their involvement in leaf-specific functions, potentially linked to the regulation of photosynthetic activity, transpiration, and responses to environmental stresses such as salt, drought, or light exposure. The significant expression of TcALKBH8 in the stem further suggests its role in stem-specific functions, which could involve processes related to vascular tissue function, such as water and nutrient transport, or possibly adaptation to mechanical stress. These findings indicate that the TcALKBH genes might have distinct functions in the physiological activities of various organs, thereby promoting the coordinated growth and development of T. chinensis. By analogy, the expression level of Arabidopsis thaliana ALKBH10B is highest in flowers, and it is involved in the floral transition process in Arabidopsis thaliana [3]. Further expression analysis under stress conditions confirmed the important role of TcALKBH genes in environmental adaptation. Under ABA treatment, the expression levels of TcALKBH genes significantly changed, especially with TcALKBH4 showing a remarkable nearly 30-fold increase in expression 6 h after ABA treatment, indicating its potential key regulatory role in the ABA-mediated signaling pathway (Figure 7A). TcALKBH6 and TcALKBH7 significantly increased at 48 h, with delayed expression, possibly because the plant may require additional time to activate the corresponding stress response pathways. The changes in TcALKBH gene expression under ABA treatment, particularly the early response of TcALKBH4 and the delayed responses of TcALKBH6 and TcALKBH7, highlight the important role of this gene family in mediating plant stress responses. The differential timing and magnitude of expression suggest that these genes may regulate different phases of stress adaptation, with some involved in immediate responses and others in longer-term survival strategies. The functional roles of these genes in T. chinensis are likely to be complex, involving the regulation of epigenetic processes that fine-tune the plant’s response to environmental stress, which aligns with similar findings in other plant species like Arabidopsis thaliana [4,5,12]. These results underscore the potential of TcALKBH genes as targets for improving the stress tolerance of T. chinensis and other salt-tolerant crops. However, the magnitude of expression changes observed in other TcALKBH genes was less pronounced compared to that reported for StALKBH genes in potato, where the expression of most StALKBH genes increased more than 10-fold during the treatment period under ABA stress [34]. These comparisons underscore the unique stress-responsive characteristics of TcALKBH genes and their potential for diverse regulatory functions in T. chinensis. In Populus, overexpression of PagALKBH9B and PagALKBH10B genes has been shown to enhance salt tolerance [20]. Since T. chinensis is a typical halophyte, analyzing the expression patterns of TcALKBH genes under salt stress helps reveal the salt tolerance mechanisms of T. chinensis. When subjected to NaCl stress, the response patterns of different TcALKBH genes showed temporal differences, suggesting a stage-specific activation mechanism (Figure 7B). The stage-specific activation could indicate the involvement of these genes in distinct phases of the plant’s stress response, with certain TcALKBH genes being upregulated early in response to stress and others later on, likely as part of a sustained adaptive response. Specifically, TcALKBH1, TcALKBH3, TcALKBH4, and TcALKBH6 underwent significant changes at 24 and 48 h, suggesting a relatively delayed response. In contrast, TcALKBH2, TcALKBH5, TcALKBH7, and TcALKBH8 began to respond significantly within 6 h, indicating that these genes may regulate salt stress through different mechanisms and at different time points, reflecting the complexity and diversity of the regulatory network involved in both early and long-term adaptation to salt stress. Under NaHCO3 stress conditions, the expression of TcALKBH1 and TcALKBH2 significantly decreased at 48 h, indicating a delayed response to NaHCO3 stress. This delayed expression pattern may reflect a slower or longer-term adaptation process, where these genes may be involved in late-stage regulatory mechanisms that help the plant adapt to sustained environmental stress. This may involve slower or longer-term adaptation processes. Although TcALKBH3, TcALKBH5, TcALKBH6, TcALKBH7, and TcALKBH8 showed significant expression changes, the magnitude of these changes was smaller, suggesting that their responses may be milder or regulated to a lesser degree, potentially participating in milder or more stage-specific stress responses. These genes may not be as heavily involved in long-term adaptation but could instead play roles in the acute or moderate stages of stress response, facilitating immediate adjustments to stressors. Overall, these findings highlight the intricate and multifaceted nature of the TcALKBH gene family’s involvement in salt stress adaptation in T. chinensis.
In conclusion, this research offers significant understanding regarding the functional variety and regulatory processes of the TcALKBH genes family in T. chinensis. The diverse expression patterns and stress response characteristics of the different TcALKBH genes suggest their pivotal role in the plant’s adaptation to salt and alkaline conditions. These findings suggest that TcALKBH genes hold promise as potential genetic targets for improving plant stress resistance in the future. Further functional validation through gene knockout or overexpression experiments will help to deeply elucidate the roles and interactions of TcALKBH genes in salt and alkaline stress adaptation.

5. Conclusions

In summary, our study has successfully identified eight TcALKBH genes from the whole genome of T. chinensis and established the basic characteristics of the TcALKBH genes family. To gain deeper insights into the regulatory functions of TcALKBH genes in the growth, development, and stress responses of T. chinensis, we analyzed their tissue-specific expression patterns and expression profiles under ABA, NaCl, and NaHCO3 stress conditions. Considering that T. chinensis is an important salt-tolerant plant and a potential pioneer species for saline–alkali lands, future research might concentrate on elucidating the regulatory functions of TcALKBH genes in response to salt–alkali stress. These findings provide invaluable foundational information for future breeding endeavors aimed at enhancing the stress tolerance of T. chinensis.

Supplementary Materials

The following supporting information can be downloaded at: https://www.mdpi.com/article/10.3390/f16030470/s1, Figure S1: qRT-PCR primer melt curves of different TcALKBH genes. Table S1: The CDS sequence of the TcALKBH genes; Table S2: Amino acid sequences used to construct evolutionary trees; Table S3: List of the motifs of TcALKBH proteins; Table S4: Ka/Ks values of TcALKBH genes pairs; Table S5: Prediction of promoter cis-regulatory elements; Table S6: Primer sequences for qRT-PCR; Table S7: The CDS sequence of TIF and α-tubulin gene.

Author Contributions

Conceptualization, Q.Y.; methodology, J.Z. and W.G.; software, J.Z. and H.Y.; validation J.Z., W.G. and H.Y.; formal analysis, J.Z.; data curation, J.Z.; writing—original draft preparation, J.Z.; writing—review and editing, J.Z., K.J. and Q.Y. All authors have read and agreed to the published version of the manuscript.

Funding

This research was funded by the Natural Science Foundation of China, grant number 32201583, and Beijing National Laboratory for Molecular Sciences, grant number BNLMS202202.

Data Availability Statement

Data are contained within the article.

Conflicts of Interest

The authors declare no conflicts of interest.

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Figure 1. Chromosomal localization of TcALKBH genes. Chromosome sizes are plotted according to relative proportions, with the left scale indicating chromosome length, and the chromosome number is indicated to the left of each chromosome.
Figure 1. Chromosomal localization of TcALKBH genes. Chromosome sizes are plotted according to relative proportions, with the left scale indicating chromosome length, and the chromosome number is indicated to the left of each chromosome.
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Figure 2. Phylogenetic analysis of TcALKBH genes in T. chinensis. Different colors represent different groups, and the TcALKBH gene is highlighted with a red asterisk. The phylogenetic tree was built using NJ (Neighbor-Joining) method, and 1000 bootstrap repeats were performed.
Figure 2. Phylogenetic analysis of TcALKBH genes in T. chinensis. Different colors represent different groups, and the TcALKBH gene is highlighted with a red asterisk. The phylogenetic tree was built using NJ (Neighbor-Joining) method, and 1000 bootstrap repeats were performed.
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Figure 3. Gene structure, conserved domain, and motif analysis of TcALKBH genes in T. chinensis. (A) The gene structure of TcALKBH, where the green boxes indicate the CDS regions and the black lines denote the intron regions. (B) Conserved domains of TcALKBH proteins analyzed by Conserved Domains Database. (C) Motif distribution of TcALKBH proteins, with different colors representing different motifs.
Figure 3. Gene structure, conserved domain, and motif analysis of TcALKBH genes in T. chinensis. (A) The gene structure of TcALKBH, where the green boxes indicate the CDS regions and the black lines denote the intron regions. (B) Conserved domains of TcALKBH proteins analyzed by Conserved Domains Database. (C) Motif distribution of TcALKBH proteins, with different colors representing different motifs.
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Figure 4. Genome-wide duplication and synteny analysis of TcALKBH genes: (A) Chromosomal distribution of duplication events. Homologous gene pairs derived from genome-wide duplication are connected by gray lines. Red lines specifically indicate segmental duplication events involving TcALKBH members. (B) Synteny analysis illustrating conserved genomic regions between T. chinensis, Arabidopsis thaliana, and Populus trichocarpa. Blue bands represent collinear genomic regions harboring ALKBH orthologs among T. chinensis, Arabidopsis thaliana, and Populus trichocarpa.
Figure 4. Genome-wide duplication and synteny analysis of TcALKBH genes: (A) Chromosomal distribution of duplication events. Homologous gene pairs derived from genome-wide duplication are connected by gray lines. Red lines specifically indicate segmental duplication events involving TcALKBH members. (B) Synteny analysis illustrating conserved genomic regions between T. chinensis, Arabidopsis thaliana, and Populus trichocarpa. Blue bands represent collinear genomic regions harboring ALKBH orthologs among T. chinensis, Arabidopsis thaliana, and Populus trichocarpa.
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Figure 5. Expression pattern of TcALKBH genes: (A) The expression pattern of TcALKBH genes in leaves, stems and roots was normalized using the expression level in leaves as the standard. An asterisk indicates a significant difference. The asterisk significance level: (* p < 0.05, *** p < 0.001, **** p < 0.0001. Student’s t-test). (B) The expression pattern of eight TcALKBH genes in the same tissue; different letters indicate significant differences. Different letters signify statistically significant variations (p < 0.05), as determined by Duncan’s test. The results are shown as the mean ± standard deviation, with three replicates (n = 3).
Figure 5. Expression pattern of TcALKBH genes: (A) The expression pattern of TcALKBH genes in leaves, stems and roots was normalized using the expression level in leaves as the standard. An asterisk indicates a significant difference. The asterisk significance level: (* p < 0.05, *** p < 0.001, **** p < 0.0001. Student’s t-test). (B) The expression pattern of eight TcALKBH genes in the same tissue; different letters indicate significant differences. Different letters signify statistically significant variations (p < 0.05), as determined by Duncan’s test. The results are shown as the mean ± standard deviation, with three replicates (n = 3).
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Figure 6. Analysis of promoter cis-acting elements of 2000 bp upstream of TcALKBH genes: (A) The promoter cis-acting elements of 2000 bp upstream of TcALKBH genes are distributed, and different colors represent different elements. (B) Classification and number of cis-acting elements of TcALKBH gene promoter. Various colors and numerals indicate the quantity of cis-acting elements.
Figure 6. Analysis of promoter cis-acting elements of 2000 bp upstream of TcALKBH genes: (A) The promoter cis-acting elements of 2000 bp upstream of TcALKBH genes are distributed, and different colors represent different elements. (B) Classification and number of cis-acting elements of TcALKBH gene promoter. Various colors and numerals indicate the quantity of cis-acting elements.
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Figure 7. Expression pattern of TcALKBH genes under stress: (A–C) the expression patterns of TcALKBH genes under ABA, NaCl, and NaHCO3 stress, respectively. The expression level of the TcALKBH gene in leaves was analyzed. The expression level of TcALKBH genes at 0 h was normalized, and asterisks represented significant differences in expression levels. The asterisk significance level: (* p < 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.0001. Student’s t-test). The results are shown as the mean ± standard deviation, with three replicates (n = 3).
Figure 7. Expression pattern of TcALKBH genes under stress: (A–C) the expression patterns of TcALKBH genes under ABA, NaCl, and NaHCO3 stress, respectively. The expression level of the TcALKBH gene in leaves was analyzed. The expression level of TcALKBH genes at 0 h was normalized, and asterisks represented significant differences in expression levels. The asterisk significance level: (* p < 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.0001. Student’s t-test). The results are shown as the mean ± standard deviation, with three replicates (n = 3).
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Table 1. Physical and chemical parameters of TcALKBH genes family members in T. chinensis.
Table 1. Physical and chemical parameters of TcALKBH genes family members in T. chinensis.
Gene IDGene NameLocusCDS (bp)Protein Length (aa)MW
(kDa)		Aliphatic IndexGravypISubcellular Localization
TC01G0348T3TcALKBH1Chr180126630.2580.56−0.3664.75nucleus
TC08G0022T1TcALKBH2Chr849816518.6775.03−0.5939.16nucleus
TC09G0148T1TcALKBH3Chr9157552459.2672.02−0.6336.49nucleus
TC10G1665T1TcALKBH4Chr10127842546.6864.49−0.5369.24nucleus
TC10G2608T1TcALKBH5Chr10155751857.4670.21−0.559.26chloroplast
TC11G0425T1TcALKBH6Chr11125741846.7180.29−0.3367.69cytosol
TC11G0725T1TcALKBH7Chr11150350056.1367.62−0.6738.19chloroplast
TC11G1777T1TcALKBH8Chr12106835539.4577.15−0.336.47nucleus
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Zhang, J.; Guo, W.; Yin, H.; Ji, K.; Yu, Q. Genome-Wide Identification and Expression Analysis of the AlkB Homolog Gene Family in Tamarix chinensis. Forests 2025, 16, 470. https://doi.org/10.3390/f16030470

AMA Style

Zhang J, Guo W, Yin H, Ji K, Yu Q. Genome-Wide Identification and Expression Analysis of the AlkB Homolog Gene Family in Tamarix chinensis. Forests. 2025; 16(3):470. https://doi.org/10.3390/f16030470

Chicago/Turabian Style

Zhang, Jingjing, Wenhui Guo, Huijuan Yin, Kongshu Ji, and Qiong Yu. 2025. "Genome-Wide Identification and Expression Analysis of the AlkB Homolog Gene Family in Tamarix chinensis" Forests 16, no. 3: 470. https://doi.org/10.3390/f16030470

APA Style

Zhang, J., Guo, W., Yin, H., Ji, K., & Yu, Q. (2025). Genome-Wide Identification and Expression Analysis of the AlkB Homolog Gene Family in Tamarix chinensis. Forests, 16(3), 470. https://doi.org/10.3390/f16030470

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