Genome-Wide Identification and Expression Analysis of AT-Hook Motif Nuclear Localized Gene Family in Birch

Abstract

The AT-hook motif nuclear localized (AHL) gene family encodes transcription factors pivotal in regulating plant growth, development, and responses to abiotic stimuli, including low temperature, salinity, darkness, and drought. In this study, we systematically identified 21 BpAHL genes in birch and characterized their sequence features, evolutionary relationships, and expression dynamics. Phylogenetic analysis classified BpAHLs into two clades (Clade-A and Clade-B) and three types (Type-I, -II, and -III), based on PPC domain and AT-hook motifs. Chromosomal mapping revealed an even distribution across nine chromosomes and one contig, with dispersed duplication events recognized as the major driver of BpAHL family expansion. Tissue-specific expression profiling uncovered striking divergence: Type-I BpAHLs displayed root-predominant expression, whereas Type-II/III BpAHLs were highly expressed in plant flowers and leaves. Notably, Type-II/III BpAHL genes in leaves showed distinct expression patterns in response to cold and heat stresses, while Type-I BpAHLs in roots were down-regulated under salt stress. This study provides a comprehensive phylogenomic and functional analysis of the AHLs in birch, providing insights into their roles in enhancing abiotic stress resilience in forest trees.

1. Introduction

With the increasing frequency of extreme weather events (e.g., droughts, floods) under climate change scenarios, understanding transcription factors (TFs)-mediated stress-responsive mechanisms in staple crops (e.g., rice, wheat) and economically important forest tree species (e.g., poplar, birch) has become crucial for ensuring global food security and sustainable forestry development [1]. Among these TFs, the AT-hook motif nuclear localized (AHL) gene family has garnered worldwide research attention because of its pivotal role in regulating plant responses to various biotic and abiotic stresses [2].

The AHL gene family was first discovered in mammalian high mobility group (HMG) proteins [3] and has subsequently been characterized in numerous terrestrial plant species with sequenced genomes, including Arabidopsis thaliana (29 members) [4], Oryza sativa (26) [5], Zea mays (37) [6], Glycine max (63) [7], Vitis vinifera (14) [8], Daucus carota (47) [9], Brassica rapa (42) [10], Populus trichocarpa (37) [11], Liriodendron chinense (21) [12], and Gossypium species (48, 51, 99) [13]. AHL proteins function as nuclear-localized transcription factors that modulate both chromosome structure assembly and target gene expression. For instance, overexpression of AHL15 can induce somatic embryogenesis without exogenous hormones and promote tetraploid formation in L. chinense, suggesting its ability to influence cell differentiation through ploidy alteration [12]. In A. thaliana, AHL13 protein interacts with poly(ADP-ribose) modification, where PARylation suppresses its DNA-binding activity, thereby fine-tuning the transcriptional regulation of some immune-related genes [14]. The DNA-binding capability of AHL protein is conferred by two conserved structural features: the AT-hook motifs and the Plants and Prokaryotes Conserved (PPC/DUF296) [4,15]. The AT-hook contains a core sequence ‘Arg-Gly-Arg-Pro’ (R-G-R-P) essential for DNA binding, while the PPC domain (~120 amino acids) features a conserved structure comprising seven β-sheets surrounding a central α-helix [4,16]. The hydrophobic C-terminal region facilitates nuclear localization and mediates protein–protein interaction, enabling AHL proteins to regulate transcription through both direct DNA binding and protein complex formation [17].

The AHL proteins in land plants are phylogenetically classified into two clades (Clade-A and Clade-B), distinguished by characteristic PPC domain signatures: ‘Leu-Arg-Ser-His’ (L-R-S-H) in Clade-A versus ‘Phe-Thr-Pro-His’ (F-T-P-H) in Clade-B [4]. These AHL proteins are further categorized into three subtypes (Type-I, -II, and -III) based on sequence variations in their AT-hook motifs. Type-I AHLs uniquely possess a C-terminal ‘Gly-Ser-Lys-Asn-Lys’ (G-S-K-N-K) extension, while both Type-II and Type-III subtypes feature a conserved ‘Arg-Lys-Tyr’ (R-K-Y) tail [4,16]. Their conserved binding capability to AT-rich DNA and expression patterns suggest AHLs have maintained critical functions throughout evolution across plant species.

The AHLs family governs pivotal biological processes in plants, encompassing three major aspects:

1.

Growth and Development Regulation.

AtAHL22 modulates flowering time and hypocotyl elongation in A. thaliana through direct regulation of FLOWERING LOCUS T (FT) and PHYTOCHROME-INTERACTING FACTOR 4 (PIF4). While its overexpression delays flowering, knockout lines exhibit enhanced hypocotyl elongation [18]. Heterotopic expression of AtAHL15 suppresses axillary meristem (AM) development while promoting apical meristem growth, thereby altering shoot architecture [19]. Additionally, AHL proteins can participate in reproductive development, including maize ear morphogenesis [20].

2.

Transcriptional Regulation.

In cotton, GhAT1 acts as a transcriptional repressor of the FSltp4 promoter in non-fiber tissues, thereby fine-tuning fiber development [21]. The soybean AT-1SNBP, which shares structural homology with HMG-A proteins, binds to the glutamine synthetase GSI5 promoter to stabilize chromatin architecture and mediate distal–proximal regulatory element interactions [22].

3.

Stress-Responsive Regulation.

Overexpression of AtAHL20 enhances immunity against bacterial pathogens in A. thaliana [23], while transgenic pepper lines overexpressing CaATL1 demonstrate broad-spectrum biotic stress resistance [24]. In rice, drought-induced OsAHL1 up-regulates OsCDPK7 expression in vascular tissues, conferring improved water-deficit tolerance [25]. Transcriptomic analyses associate specific GmAHLs with drought and flooding responses in soybean, underscoring their stress-responsive regulatory functions [7].

Despite these advances, research on AHLs in perennial woody plants remains limited. Birch (Betula platyphylla Suk.), a keystone broad-leaved deciduous hardwood tree species, is widely distributed across northern Europe, Russia, and northern China, where it forms both pure stands and mixed forests [26]. Birch holds significant medicinal, commercial, and ecological values, e.g., betulin—a bioactive compound extracted from its bark—exhibits cytotoxic activity with potential anticancer applications [27,28]. Its high-density wood, prized for its attractive white grain, is extensively used in furniture manufacturing, construction, and paper production [29]. Ecologically, sustainable birch cultivation enhances vegetation coverage, mitigates soil erosion, improves water conservation, and maintains forest ecosystem balance. However, natural birch are highly vulnerable to abiotic stresses, including salinity [30], osmotic stress [31], and low-temperatures [32]. Moreover, the AHL gene family in birch has not yet been comprehensively characterized. Here, we systematically investigated the birch AHL gene family through bioinformatics identification, phylogenetic and evolution analysis, structural predictions, expression profiling, and abiotic stress response assays. Our findings deepen the understanding of AHL gene functions in forest trees and provide a foundation for their application in breeding and stress resilience engineering.

2. Materials and Methods

2.1. Plant Material and Treatments

The seeds of Betula platyphylla used in this study were derived from ‘DL-1’, a superior white birch strain characterized by high differentiation efficiency, rapid growth, and elevated biomass [33]. These seeds were obtained from the State Key Laboratory of Tree Genetics and Breeding (TGB) at Northeast Forestry University in 2020. For germination, surface-sterilized seeds were cultured on Hoagland’s solution in Petri dishes for one week. Germinated seedlings were then transferred to plastic pots containing a sterilized growth medium (peat moss: garden soil: river sand = 5:2:1, v/v/v) and maintained at 26 °C under a 14 h light/10 h dark photoperiod [28]. After three months, various tissues (leaves, stems, and roots) were collected in three independent biological replicates per sample, flash-frozen in liquid nitrogen, and stored at −80 °C for qRT-PCR experiments.

To induce drought stress, three-month-old seedlings of uniform growth status were irrigated with 0.2 mol/L mannitol solution. Whole plants were photographed at 9:00 a.m. on days 1, 3, and 5 post-treatment in a PERCIVAL growth chamber (26 °C, ~40% relative humidity). Simultaneously, root tissues were independently collected (n = 3 biological replicates per group), immediately flash-frozen in liquid nitrogen, and stored at −80 °C for qRT-PCR experiments.

2.2. Characterization of BpAHL Gene Family

Putative AHL proteins in the birch genome [34] were identified using the HMMER 3.0 program [35] with the PPC/DUF296 (PF03479) domain (https://pfam.xfam.org; accessed on 2 January 2024) [36,37], and further validated via NCBI-BLASTp (E-value ≤ 1 × 10⁻¹⁰). Domain architecture was confirmed using Conserved Domains Database (CDD) (https://www.ncbi.nlm.nih.gov/Structure/cdd/wrpsb.cgi; accessed on 4 January 2024) [38] and SMART (https://smart.embl.de; accessed on 5 January 2024) [39], with an E-value cutoff of 1 × 10⁻⁵. Only proteins containing both one PPC domain and at least one AT-hook motif were classified as BpAHL family members; these were systematically numbered according to genomic position.

Chromosomal localization, gene length, CDS length, protein length, and GO annotation of the BpAHL gene family were extracted from birch genome annotations (https://genomevolution.org/coge/GenomeInfo.pl?gid=35080; accessed on 1 July 2023) [34]. The molecular weight (MW) and theoretical isoelectric point (pI) of each BpAHL protein were computed using ExPASy Proteomics Server (https://www.expasy.org; accessed on 20 January 2024) [40], and subcellular localization was predicted via the Plant-PLoc website (http://www.csbio.sjtu.edu.cn/bioinf/plant; accessed on 25 January 2024) [41].

Tertiary structures of the BpAHL proteins were modeled via SWISS-MODEL (https://swissmodel.expasy.org/interactive; accessed on 3 February 2024) [42], with output PDB files visualized in Swiss PDB Viewer v4.0.1 (https://spdbv.unil.ch; accessed on 5 February 2024) [43]. For biological function, GO enrichment analysis was performed with goatools v0.8.2 (https://github.com/tanghaibao/goatools; accessed on 10 February 2024) [44], where significantly enriched terms (Fisher’s exact test, p-value ≤ 0.05) were identified.

2.3. Phylogenetic Analysis of BpAHL Protein Family

To elucidate the evolutionary relationships of AHL proteins across diverse plant species, we conducted a comprehensive phylogenetic analysis with maize (https://plants.ensembl.org/index.html; accessed on 10 July 2023, assembly: Zm-B73-REFERENCE-NAM-5.0), soybean (https://phytozome-next.jgi.doe.gov; accessed on 10 July 2023, assembly: Glycine max Wm82.a2.v1), sorghum (https://plants.ensembl.org/index.html; accessed on 10 July 2023, assembly: Sorghum_bicolor_NCBIv3), A. thaliana (https://www.arabidopsis.org; accessed on 10 July 2023, assembly: TAIR10), and birch. The phylogenetic tree was constructed with MEGA11 (https://www.megasoftware.net; accessed on 12 July 2023) [45], which involved three key steps: (1) multiple sequence alignment with MUSCLE algorithm, (2) tree construction using the neighbor-joining (NJ) method, and (3) evaluation of branch support with 1000 bootstrap replicates to ensure topological reliability. The phylogenetic tree was exported in Newick format and visualized using the Interactive Tree of Life (iTOL) platform [46], with all protein sequences and the corresponding Newick file provided in Tables S1 and S2.

2.4. Protein Domain, Gene Structure, and Motif Prediction of BpAHL Gene Family

The BpAHL protein sequences were initially analyzed using the SMART website to identify conserved amino acid residues. Following our established methodology [7], we recorded specific residues as domain identifiers: the first 4/7 and 8/10 residues of the N-terminal and C-terminal AT-hook motifs, respectively, and the first 4 residues of the PPC domain. Phylogenetic classification of BpAHL proteins was performed using MEGA11 software, adopting an approach consistent with our previous work [7]. Genomic features including coding sequences (CDSs), exons, and introns of BpAHL genes were extracted from genome annotation files. These structural elements were visualized using the ‘Gene Structure View’ module in TBtools v2.136 (https://github.com/CJ-Chen/TBtools-II/releases; accessed on 17 October 2023) [47], enabling clear representation of gene architectures. Conserved protein motifs were predicted using the MEME website (https://meme-suite.org/meme; accessed on 15 February 2024) [48], with a stringent E-value cutoff of 1 × 10⁻⁵. The ten most statistically significant motifs were identified and graphically represented using the ‘Visualize MEME Motif Pattern’ module in TBtools software, facilitating comparative analysis of motif distributions across BpAHL proteins.

2.5. Chromosomal Localization, Gene Duplication Events, Synteny and Ka/Ks Analysis of BpAHL Gene Family

The chromosomal locations of BpAHL genes were mapped and visualized using the ‘Gene Location Visualize’ module in TBtools. Gene duplication events were identified by analyzing BpAHL proteins with MCScanX (https://github.com/wyp1125/MCScanX; accessed on 27 February 2024) [49], using an E-value threshold of 1 × 10⁻⁵, with numerical identifiers representing duplication types: ‘0’ (singleton), ‘1’ (dispersed), ‘2’ (proximal), ‘3’ (tandem), and ‘4’ (WGD/segmental). Synteny analysis of AHL proteins between five species (A. thaliana, soybean, rice, maize, Populus) and birch was performed using the ‘One Step MCScanX’ module in TBtools, with comparative genomic data obtained from the Ensembl Plants, Phytozome and TAIR websites. The evolutionary constraints on duplicated gene pairs (type ‘4’) were evaluated by calculating synonymous (Ks) and non-synonymous (Ka) substitution rates and their ratios (Ka/Ks) using the ‘Simple Ka/Ks Calculator’ module in TBtools. Divergence times were estimated using the formula T = Ks/2r, where r represents the clock-like rate of divergence. For dicots, r is typically estimated at 1.5 × 10⁻⁸ substitutions per synonymous site per year [50].

2.6. Cis-Acting Regulatory Elements (CREs) in the Promoters of BpAHL Genes Family

The 2.0 kb upstream promoter regions of BpAHL genes were extracted from the birch genome using the ‘Gtf/Gff3 Sequences Extract’ module in TBtools, followed by comprehensive CREs prediction via PlantCARE (https://bioinformatics.psb.ugent.be/webtools/plantcare/html; accessed on 8 March 2024) [51]. The analysis focused on identifying and classifying the ten most abundant CREs, which were subsequently visualized using the ‘Simple BioSequence Viewer’ module in TBtools for comparative analysis of promoter architectures.

2.7. Tissue-Specific Transcriptome Analysis of BpAHL Gene Family

Illumina high-throughput sequencing data encompassing multiple tissue types (leaf, root, flower, and xylem) from two-year-old birch specimens were obtained from the Experimental Station of Northeast Forestry University [52] and downloaded from the NCBI Sequence Read Archive (PRJNA535361). For expression quantification, clean reads were aligned to the birch genome using HISAT2 (https://daehwankimlab.github.io/hisat2; accessed on 2 April 2024) [53], followed by quantification with StringTie (https://ccb.jhu.edu/software/stringtie; accessed on 6 April 2024) [54], with default parameters, to generate transcripts per million (TPM) values. This approach employs probabilistic read allocation to transcript models, preserving multi-mapping reads to more accurately represent transcript abundance and minimize quantification bias. Finally, BpAHL expression profiles were row-scaled and visualized as a heatmap using the ‘pheatmap’ function in R (https://www.r-project.org; accessed on 15 August 2023).

2.8. Abiotic Transcriptome Analysis of BpAHL Gene Family

Transcriptome datasets were analyzed to examine BpAHL gene expression profiling under diverse abiotic stress conditions in Betula platyphylla. Four distinct experimental datasets were obtained from the NCBI Sequence Read Archive (SRA):

1.

Low-Temperature Treatments in Short Term.

Transcriptome data (PRJNA532995) from leaves of two-month-old birch treated with low-temperature (6 °C) for six periods (0.5 h, 1.0 h, 1.5 h, 2.0 h, 2.5 h, and 3.0 h) [55].

2.

Low- and High-Temperature Treatments in Long Term.

Transcriptome data (PRJNA811313) from leaves of two-month-old birch treated with low-temperature (6 °C) and high-temperature (35 °C) for six periods (6.0 h, 1 d, 2 d, 4 d, 7 d, and 14 d) [56].

3.

Salt Stress Treatments.

Transcriptome data (PRJNA790472) from leaves and roots of 10-week-old birch treated with 24 h of 0.2 M NaCl condition [57].

4.

Light/Dark Treatments.

Transcriptome data (PRJNA759706) from leaves of one-month-old birch treated with four days of light and dark cycles [58].

All datasets were processed using the bioinformatics pipelines described in Section 2.7, with genes showing TPM values < 2.0 excluded from subsequent analyses.

2.9. RNA Extraction and Quantitative RT-PCR (qRT-PCR)

Total RNA was isolated from fresh leaves, stems and roots of three-month-old birch seedlings using the OminiPlant RNA Kit (DNase I). High-quality RNA was reverse-transcribed into cDNA using the PrimeScripTM RT Reagent Kit with gDNA Eraser in a two-step process:

1.

Genomic DNA elimination.

A total of 10 µL of reaction mixture containing 2 µL RNA (500 ng/μL), 2 µL 5× gDNA Eraser Buffer, 1 µL gDNA Eraser, and 5 µL RNase-free ddH₂O was incubated at 42 °C for 2 min followed by 10 min on ice.

2.

cDNA synthesis.

The reaction mixture was brought to 20 µL by adding 1 µL PrimeScript™ RT Enzyme Mix I, 4 µL 5× PrimeScript™ Buffer2, 1 µL RT Primer Mix, and 4 µL RNase-free ddH₂O, and then incubated at 37 °C for 15 min, 85 °C for 5 s, and 4 °C for 10 min. The synthesized cDNA was diluted 20-fold and stored at −40 °C.

For qRT-PCR, the reaction mixtures (15 µL) contained 6.3 µL diluted cDNA, 0.6 µL each of forward and reverse primers (Table S3), and 7.5 µL FastStart Universal SYBR Green Master. Amplification was performed on an ABI7500 instrument (Applied Biosystems) under the following conditions: 95 °C for 10 min; 40 cycles of 95 °C for 10 s, and 60 °C for 30 s. This was followed by melt curve analysis (95 °C for 1 min, 65 °C for 30 s, then 95 °C for 0.11 °C per second down to 5 °C). Technical triplicates were performed with each sample, with relative transcript levels calculated using the 2^−ΔΔCt method [59] and normalized to tubulin as the internal control [60,61].

2.10. Statistical Analysis

Differences in relative gene expression levels resulting from qRT-PCR were evaluated using one-way ANOVA. For transcriptome-derived gene expression levels (TPMs), differences were assessed using Student’s t-test. Statistical significance was set at a threshold of p-value ≤ 0.05. All figures were generated using the R program (https://www.r-project.org; accessed on 15 August 2023).

3. Results

3.1. Identification of BpAHL Gene Family

In this study, we identified 21 putative AHL genes in birch using the Hidden Markov Model (HMM) engine, systematically designated as BpAHL01 to BpAHL21 (

Table 1. Genomic and chemical characteristics of the 21 BpAHLs, including gene name, gene accession, genomic location, type, gene length, CDS length, protein length, molecular weight (MW), isoelectric point (pI), and subcellular localization. The designations ‘C’, ‘Cm’, and ‘N’ correspond to ‘Chloroplast’, ‘Cell membrane’, and ‘Nucleus’, respectively.

Gene Name	Gene Accession	Genomic Location	Type	Gene Length (bp)	CDS Length (bp)	Protein Length (aa)	Molecular Weight (Da)	Isoelectric Point	Subcellular Localization
BpAHL01	Bpev01.c0774.g0008	Chr1: 11,387,186–11,387,992	I	807	807	268	27,489	5.97	N
BpAHL02	Bpev01.c0616.g0003	Chr2: 12,611,347–12,618,513	III	7167	1041	346	35,800	8.97	Cm/C/N
BpAHL03	Bpev01.c0362.g0004	Chr2: 19,921,747–19,922,535	I	789	789	262	27,321	8.38	C/N
BpAHL04	Bpev01.c1484.g0011	Chr3: 16,791,132–16,791,854	I	723	723	240	24,150	8.97	N
BpAHL05	Bpev01.c1390.g0007	Chr4: 10,015,299–10,026,351	III	11,053	1038	345	35,560	9.00	C/N
BpAHL06	Bpev01.c1390.g0016	Chr4:10,121,683–10,122,537	I	855	855	284	29,385	5.48	N
BpAHL07	Bpev01.c0365.g0010	Chr5: 2,456,997–2,464,503	I	7507	1086	361	36,878	4.73	N
BpAHL08	Bpev01.c0540.g0008	Chr5: 6,984,368–6,996,557	III	12,190	741	246	25,202	9.73	N
BpAHL09	Bpev01.c0027.g0117	Chr5: 23,300,208–23,306,294	II	6087	1020	339	34,883	10.05	N
BpAHL10	Bpev01.c0874.g0035	Chr5: 26,805,688–26,806,626	I	939	939	312	32,923	7.16	N
BpAHL11	Bpev01.c0169.g0027	Chr8: 22,673,422–22,678,438	II	5017	1095	364	37,421	8.51	N
BpAHL12	Bpev01.c0480.g0053	Chr9: 391,896–392,816	I	921	921	306	30,838	7.93	N
BpAHL13	Bpev01.c0480.g0043	Chr9: 485,086–490,203	II	5118	1122	373	37,101	10.08	N
BpAHL14	Bpev01.c0902.g0012	Chr13: 7,069,248–7,078,300	II	9053	972	323	33,737	5.68	C
BpAHL15	Bpev01.c0223.g0044	Chr13: 10,271,229–10,272,056	I	828	828	275	28,092	7.05	N
BpAHL16	Bpev01.c1544.g0004	Chr14: 2,458,829–2,467,966	III	9138	1452	483	52,128	5.64	C/N
BpAHL17	Bpev01.c1544.g0009	Chr14: 2,534,436–2,535,362	I	927	927	308	33,002	6.53	N
BpAHL18	Bpev01.c0449.g0016	Chr14: 4,176,010–4,181,875	II	5866	1032	343	35,228	10.25	Cm/C/N
BpAHL19	Bpev01.c0964.g0005	Chr14: 15,052,985–15,053,881	I	897	897	298	30,770	6.15	N
BpAHL20	Bpev01.c0964.g0003	Chr14: 15,082,749–15,091,115	II	8367	1119	372	37,807	9.57	N
BpAHL21	Bpev01.c1465.g0006	Contig1465: 35,258–36,043	I	786	786	261	27,447	6.06	N

). Tertiary structure analysis confirmed that all candidate proteins contain conserved architectural features, including PPC/DUF296 domains and AT-hook motifs (Figure S1 and Table S4), validating their classification as AHL family members. The BpAHL proteins exhibited molecular weights ranging from 24,150 Da (BpAHL04) to 52,128 Da (BpAHL16), with isoelectric points (pI) varying between 4.73 (BpAHL07) and 10.02 (BpAHL18), averaging 7.7. Subcellular localization predictions indicated exclusive nuclear targeting for all BpAHL proteins (Table 1), consistent with their functional role as transcription factors in nuclear gene regulation.

3.2. Phylogenetic Analysis of BpAHL Protein Family

To infer the evolutionary relationships among the AHL proteins in birch, we conducted a phylogenetic analysis using full-length BpAHL protein sequences. Our results indicated that BpAHL proteins are grouped into two major clades: Clade-A (11 members, 52%), and Clade-B (10, 48%) (Figure 1). Further protein sequence alignments subdivided these clades into three types: Type-I (11, 52%), Type-II (6, 29%), and Type-III (4, 19%) (Figure 1 and Figures S2–S4). This mirrors the classification patterns observed in other land plants such as maize, rice, and soybean [4,5,6,7,13].

Comparative sequence analysis uncovered distinct conservation patterns in the functional domains. The PPC domain showed greater variability in Clade-A (‘L-R-S-H’ in 45% and ‘L-R-A-H’ in 27% of members) compared to the predominant ‘F-T-P-H’ motif (80%) in Clade-B (Figure 1 and Figures S2–S4). While N-terminal AT-hook motifs were highly conserved (‘R-G-R-P’ in Clade-A; ‘R-G-R-P-R-K-Y’ in Clade-B), C-terminal motifs displayed significant divergence, with Type-I members containing ‘G-S-K-N’ (73%) and Types II-III lacking this motif entirely. Taken together, the sequence diversity of AT-hook motifs and the PPC domain observed in the BpAHL proteins likely contributes to a varied array of biological functions.

Reconstruction of phylogenetic relationships across five species (A. thaliana, soybean, maize, sorghum, and birch) confirmed the universal conservation of Clade-A and Clade-B, though with species-specific expansion patterns (Figure 2). Specifically, there are 15, 34, 20, 14, and 11 AHL proteins in A. thaliana, soybean, maize, sorghum, and birch, respectively, belonging to Clade-A, while Clade-B includes 14, 29, 17, 11, and 10 proteins from these species. Notably, birch contained the fewest AHL members (11 in Clade-A, 10 in Clade-B) compared to the expanded families in soybean (34/29) and maize (20/17). This conserved clade structure supports previous findings and an ancient divergence event [4], while the limited paralog expansion in birch highlights potential functional optimization within this woody species.

3.3. Sequence Characterization of BpAHL Gene Family

Comparative analysis of BpAHL gene structures revealed distinct organizational patterns among BpAHL types (Figure 3A and Table 1). Type-I members predominantly (10/11) feature single-exon structures (723–939 bp), while BpAHL07 represents an exception with two exons (7507 bp). In contrast, Type-II/III members exhibit more complex architectures, containing 5–8 exons within substantially longer genes (5017–12,190 bp), suggesting evolutionary expansion from simpler Type-I ancestors—a pattern consistent with maize AHL evolution [6]. This structural complexity, particularly through intron acquisition, likely facilitates alternative splicing and functional diversification in higher plants.

Subsequently, we compared the motifs among BpAHL proteins (Figure 3B). The ten most frequently conserved motifs are shown, with the corresponding amino acid residues illustrated in Figure S5. The lengths of these motifs ranged from 8 to 41 amino acids, with site ranges varying from 3 to 21 (Table S5). As expected, the 4th to 6th amino acid residues of Motif3, ‘R-G-R-P’, and the 5th to 11th amino acid residues of Motif5, ‘R-G-R-P-R-K-Y’, both represent conserved residues in N-terminal AT-hook motifs, which are predominantly found in Type-I (11 members, 100%) and Type-II/III (10, 100%) BpAHLs, respectively. The 9th to 16th amino acid residues of Motif3, ‘G-S-K-N-K-P-K-P’, as conserved residues in C-terminal AT-hook motifs, were also exclusively present in Type-I (11, 100%). Notably, both conserved amino acid residues at C- and N-terminals of the AT-hook motifs share the same core, ‘R-G-R-P’, indicating their role in binding to the minor groove of AT-rich B-form DNA for regulatory functions [62]. While Motif1, Motif2, Motif4, and Motif6 were universally conserved, Motif7 appeared to be specific to Type-I, suggesting both functional conservation and type-specific specialization within the BpAHL protein family.

3.4. Chromosome Locations of BpAHL Gene Family

Genomic mapping revealed that 20 of the 21 identified BpAHL genes are distributed across nine birch chromosomes (1, 2, 3, 4, 5, 8, 9, 13, and 14), with chromosome 14 harboring the highest number (five genes) followed by chromosome 5 (four genes) (Figure 4A). The remaining chromosomes showed variable distributions: chromosomes 2, 4, 9, and 13 each contained two BpAHL genes, while chromosomes 1, 3, and 8 each possessed a single copy. The exceptional BpAHL21 was localized to an unanchored genomic fragment (Contig1465), suggesting it may reside in an unassembled chromosomal region.

3.5. Gene Functions of BpAHL Gene Family

To elucidate the biological functions of BpAHLs, we conducted gene ontology (GO) enrichment analysis across three aspects: biological process (BP), cellular component (CC), and molecular function (MF) (Figure 4B, Table S6). In terms of the MF, BpAHLs exhibited significant enrichment in DNA binding (GO:0003677, p-value = 3.5 × 10⁻¹¹) and adenine-thymine-rich DNA binding (GO:0003680, p-value = 3.2 × 10⁻¹⁰), reinforcing their role in regulating AT-rich DNA sequences. Regarding BPs, BpAHLs were primarily engaged in metabolic and biosynthetic processes, including the brassinosteroid metabolic process (GO:0016132, p-value = 4.58 × 10⁻⁶). Considering CCs, BpAHLs were primarily localized in the nucleus (GO:0005730, p-value = 2.2 × 10⁻³) and nucleoplasm (GO:0005654, p-value = 1.6 × 10⁻⁴), suggesting their involvement in nuclear activities. Collectively, the systematic GO enrichment offers a comprehensive understanding of genes’ biological roles, facilitating further exploration of their mechanisms and potential significance in organisms.

3.6. Evolutionary Analysis of BpAHL Gene Family

Gene duplication events, arising from region-specific duplication or polyploidization, play a crucial role in gene family expansion and plant genome evolution [63,64]. Here, we investigated duplication patterns in the BpAHL gene family and found that ~81% (17 members) originated from dispersed duplication, while ~19% (4) resulted from segmental duplication (Figure 4C). Surprisingly, no tandem duplications were detected among the BpAHLs (Table S7), suggesting that BpAHLs have never undergone tandem duplication during birch long-term evolution.

Furthermore, we computed Ka/Ks ratios for those segmental duplication gene pairs, finding that BpAHL11/BpAHL14 and BpAHL09/BpAHL18 exhibited ratios of 0.33 and 0.27, respectively, both significantly below 1, indicating strong purifying selection during their evolutionary history. The estimated divergence times for these pairs were ~48.5 and ~36.4 million years ago (MYA), aligning with the evolutionary timeline of Populus trichocarpa AHLs (1.36–58.62 MYA) but substantially later than most AHLs in rice (13.82–173.89 MYA) [11,65].

To investigate evolutionary relationships, we assessed collinearity between BpAHLs and AHL proteins from two monocots (rice, maize) and three dicots (A. thaliana, soybean, Populus) (Figure 4D). Notably, 8 (~38%), 7 (~33%), 12 (~57%), 15 (~71%), and 16 (~76%) BpAHL members showed high homology with the AHLs from these species, respectively. These results support earlier findings in birch [60] and highlight the evolutionary divergence between monocot and dicot AHLs.

3.7. Cis-Acting Regulatory Elements in Promoters of BpAHL Gene Family

In organisms, promoter regions located upstream of the genes that bind to transcription factors (TFs) are called cis-acting regulatory elements (CREs) and encode blueprints for proper spatiotemporal patterning of gene expression and responses to environmental stimuli [66,67]. Here, we predicted ten classes of CREs in BpAHL promoters, associated with hormones (gibberellin, GA; methyl jasmonate, MeJA; abscisic acid, ABA; auxin; and salicylic acid, SA) and abiotic stress responses (light response; anaerobic induction; low-temperature response; and drought response) (Figure 5A, Table S8). The predicted CREs were present in 7 to 21 BpAHL genes, with each promoter containing three to eight distinct CREs (Figure 5B–D). The abiotic stress-related CREs, particularly light-responsive and anaerobic induction CREs, showed the broadest distribution, appearing in 21 and 19 BpAHLs, respectively. Hormone-responsive CREs (ABA, GA, and MeJA) were detected in 11 to 13 BpAHLs, while SA-responsive and low-temperature-responsive CREs exhibited more limited distributions, present in 7–8 BpAHLs. Notably, BpAHL05, BpAHL08, and BpAHL17 contained the most comprehensive set of CREs, with all eight types equally represented between hormone (four classes) and abiotic stress (four classes) responses (Figure 5B–D). These findings suggest that diverse CREs enable BpAHL genes to potentially respond to various environmental and hormonal signals.

3.8. Transcriptome Profiles of BpAHL Gene Family in Different Plant Tissues

To gain insights into potential functions of BpAHL genes in plant growth and development, we analyzed their gene expression patterns in four tissues of two-year-old birch using previously reported transcriptome data [52]. The 21 BpAHL genes showed predominant expression in flowers, leaves, and roots, displaying two distinct expression profiles (Figure 6A).

Specifically, the nine BpAHL genes (BpAHL11, BpAHL20, BpAHL03, BpAHL08, BpAHL13, BpAHL14, BpAHL18, BpAHL05, and BpAHL15) exhibited significantly higher expression in flowers and leaves compared to roots and xylem. Notably, ~78% (7/9) of these belonged to Type-II/III BpAHLs (Figure 6A, Table S9). In contrast, the remaining 11 genes (BpAHL02, BpAHL16, BpAHL12, BpAHL06, BpAHL19, BpAHL21, BpAHL07, BpAHL04, BpAHL10, BpAHL01, and BpAHL17) showed preferential root expression, with ~81% (9/11) classified as Type-I. In addition, the expression patterns were further validated by qRT-PCR experiments of randomly selected BpAHLs (Figure 6B). The distinct demarcation in expression patterns between BpAHL types (Type-I versus Type-II/III) across various tissues—the belowground versus the aboveground—implies that different types of BpAHLs may fulfill diverse biological functions in the tissues of birch.

3.9. Transcriptome Analysis of BpAHL Gene Family in Response to Abiotic Stress

To investigate the responses of the BpAHL gene family to various abiotic stresses, we performed a transcriptome analysis based on a series of artificial treatments guided by predicted CREs in promoters of BpAHLs (Figure 5). The treatments included temperature extremes (6 °C and 35 °C), salt stress (0.2 M NaCl for 24 h), and light/dark cycles (4 days). We systematically examined expression patterns of all 21 BpAHL genes under each condition (Figure 7).

3.9.1. Type-I BpAHLs Show a Limited Cold Response, While Type-II/III Show a Dynamic Response

Under low-temperature (6 °C) conditions, nearly all (10/11) Type-I BpAHLs (BpAHL01/03/04/06/10/12/15/17/19/21) exhibited low expression levels and no obvious responses in leaves (Table S10). Contrastingly, five Type-II (BpAHL09/11/13/18/20) and four Type-III (BpAHL02/05/08/16) BpAHLs, along with Type-I BpAHL07, displayed dynamic responses, revealing two distinct expression patterns (Figure 7A, Table S10). Specifically, six BpAHLs of Type-II/III (BpAHL02/08/11/13/18/20) showed progressive down-regulation during cold exposure (Figure 7A); conversely, four genes (BpAHL05/07/09/16) peaked at 1 h treatment. Extended cold exposure revealed further complexity: BpAHL18/20 showed early induction in the first 4 days, and BpAHL05/13 peaked on days 1–2, while BpAHL14/15 exhibited delayed responses, with BpAHL14 peaking on day 4 and BpAHL15 declining progressively (Figure 7B, Table S11).

3.9.2. Almost BpAHLs Show Down-Regulation Under Heat Stress on the First Day

Under high-temperature (35 °C) conditions, the expression patterns of BpAHLs were strikingly similar (Figure 7C, Table S12). With the exception of BpAHL12, BpAHL05, and BpAHL07, the expression levels of the other 10 BpAHLs dropped to their lowest values on the first day before gradually rebounding. Collectively, these results indicate that primarily Type-II/III BpAHLs respond to both low- and high-temperature stresses in leaves, with their varying expression patterns suggesting different biological functions in temperature stress responses.

3.9.3. Almost BpAHLs Down-Regulated Under Salt Stress in Root Tissue

Only 24% of BpAHLs (five members) responded to salt stress in leaves, predominantly Type-II/III (BpAHL05/08/09/11) and Type-I BpAHL07 (Figure 7D, Table S13). Two BpAHLs were up-regulated while three showed down-regulation, indicating specialized roles in leaf salt tolerance. In roots, 71% of BpAHLs (15 members) responded to salt stress, with 80% (12 members) showing significant down-regulation (Figure 7E, Table S14). This included six Type-I (BpAHL01/06/07/12/19/21) and six Type-II/III (BpAHL02/05/08/09/18) genes, revealing tissue-specific regulatory networks for salt adaptation.

3.9.4. Only Type-II/III BpAHLs Respond to Light/Dark Cycles

Approximately 24% of BpAHLs (all Type-II/III: BpAHL02/05/08/09/20) responded significantly to light/dark cycles in leaves (Figure 7F, Table S15), suggesting their specialized role in photoperiod-related processes.

3.10. Expression Analysis of BpAHL Genes Under Drought Treatment

Previous research has established that AHL family proteins (AT-hook motif nuclear localized proteins containing ankyrin repeats) play crucial regulatory roles in Oryza sativa under drought stress responses [25]. In our drought stress experiments with birch (Betula platyphylla), visible phenotypic alterations were observed (Figure 8A). Following 5–6 days of progressive drought treatment, stressed individuals exhibited marked growth retardation and wilting symptoms compared to well-watered controls, although the phenotypic differences remained relatively moderate during the initial period. Intriguingly, qRT-PCR experiments revealed a significant up-regulation of gene expression in root tissues under drought stresses (Figure 8B), suggesting their potential involvement in coordinating root-specific adaptive responses to these contrasting abiotic stresses.

4. Discussion

The AT-hook motif nuclear localized (AHL) gene family has been well documented to play pivotal roles in transcriptional regulation, plant growth and development, and stress responses [15,17,68]. Extensive systematic research has been conducted on this gene family across a variety of terrestrial plant species, encompassing dicotyledonous herbs such as A. thaliana (29 members) [4], Glycine max (63) [7], Daucus carota (47) [9], Brassica rapa (42) [10], and Gossypium species (48–99) [13]; monocotyledonous herbs like Oryza sativa (26) [5], and Zea mays (37) [6]; and woody plants including Vitis vinifera (14) [8], Populus trichocarpa (37) [11], and Liriodendron chinense (21) [12]. Here, we identified 21 AHL members in birch (Figure 1 and Table 1), representing the second-lowest number among characterized species, with only grape having a lower number. Notably, the number of AHL members is generally lower in woody plants compared to herbs, suggesting that the AHL gene family might fulfill indispensable functions throughout the entire lifecycle of trees.

Phylogenetic analysis classified the BpAHL proteins into two major clades (Clade-A and Clade-B) based on their PPC domain sequences—Clade-A harbors the conserved ‘L-R-S-H’ motif, while Clade-B contains the ‘F-T-P-H’ motif. Further subdivision based on AT-hook motifs categorized the members into three types: Type-I (with the ‘R-G-R-P’ motif at the N-terminus), Type-II (with ‘R-G-R-P’ at the C-terminus), and Type-III (lacking this conserved C-terminal residues) (Figure 1). These two functional domains are essential for nuclear localization and transcriptional regulation of AHL proteins [15,37,69,70]. Notably, mutations in the AT-hook motif could impair chromatin-binding activity [71].

Notably, distinct differences were present in sequence characteristics and physicochemical properties between Type-I and Type-II/III BpAHLs. Specifically, except for BpAHL07, all other Type-I BpAHLs are shorter than 1.0 kb, whereas Type-II/III BpAHLs exceed 5.0 kb in length (Figure 3A and Table 1). However, the variation in CDS lengths—particularly protein lengths—is relatively minor. This observation suggests that, although Type-II/III BpAHLs contain significantly more CDSs than Type-I BpAHLs, their translated proteins remain similar in size. This phenomenon may result from evolutionary processes such as genome duplication, transposon insertion, non-neutral selection, and co-evolving residues [72]. Additionally, these findings support the hypothesis that Type-II/III BpAHLs originated from Type-I BpAHLs and may employ a greater number of introns to generate novel splice variants, enabling precise regulation of gene function in response to specific developmental stages and tissue types [4,73].

Comparative analysis of molecular weight (MW), isoelectric point (pI), and subcellular localization among the BpAHL proteins revealed distinct patterns (Table 1). Type-I BpAHLs exhibited smaller molecular weights (24,150–36,878 Da; average ~29,845 Da) compared to Type-II (33,737–37,807 Da; average ~36,030 Da) and Type-III (25,202–52,128 Da; average ~37,173 Da). Similarly, Type-I proteins showed slightly lower pI values (4.73–8.97; average ~6.80) than Type-II (5.68–10.25; average ~9.00) and Type-III (5.64–9.73; average ~8.30), suggesting potential functional specialization in different micro-environments. Subcellular localization predictions demonstrated that all Type-I BpAHLs (11 proteins, 100%) were nuclear-localized, while half of Type-II/III BpAHLs (5 members) showed additional chloroplast localization. This finding raises the intriguing possibility that some BpAHL proteins may exert regulatory functions through chloroplast DNA binding [12]. Together, these results reinforce the relative conservation of Type-I BpAHL properties, in contrast to the greater variability observed in Type-II/III members.

Chromosomal distribution analysis revealed that these 21 BpAHL genes are relatively evenly distributed across birch’s nine chromosomes and one contig (Figure 4A), showing no significant correlation with chromosome length (Spearman correlation, p-value > 0.05). Gene duplication analysis indicated that most BpAHLs (17/21) arose from dispersed duplication events (Figure 4C), a pattern similar to maize but distinct from soybean [6,7]. This difference likely reflects soybean’s status as an ancient tetraploid [74], in contrast to birch, which lacks evidence of whole-genome duplication (WGD) in its evolutionary history [34]. This evolutionary distinction might also account for the relatively small BpAHL gene family in birch. Comparative synteny analysis demonstrated strong conservation between birch (a dicot) and other dicots (Glycine max, Populus trichocarpa, and A. thaliana), but weaker synteny with monocots (Oryza sativa and Zea mays) (Figure 4D). This divergence may result from mutated Type-I BpAHLs in monocots [6], highlighting an important evolutionary differentiation in the AHL gene family between monocotyledonous and dicotyledonous plants.

The AHL gene family displayed conserved tissue-specific expression patterns across various plant species, with soybean AHLs predominantly expressed in roots, meristems and epicotyls [7], cabbage AHLs in roots and buds [10], and maize AHLs in roots, embryos, endosperm and seeds [6]. Here, this study reveals that birch AHLs exhibited similar type-dependent tissue specificity: Type-I BpAHLs show root-predominant expression (below-ground tissues), while Type-II/III members exhibit preferential expression in flowers and leaves (above-ground tissues) (Figure 6A and Table S9), a pattern confirmed by qRT-PCR experiments of BpAHL06, BpAHL21 and BpAHL09 (Figure 6B). This expression dichotomy suggests functional specialization, with Type-I genes likely involved in root development and nutrient uptake (consistent with Arabidopsis AtAHL18/AtAHL29 [75,76]), whereas Type-II/III genes might participate in aerial tissue processes like photosynthesis, flowering or defense mechanisms [4,5,6].

The CREs within gene promoters play crucial roles in modulating transcriptional networks governing abiotic stimulus responses, hormone signaling, and developmental processes [77]. Previous research has demonstrated this regulatory function in AHL family members: AtAHL15 and other Clade-A proteins directly regulate downstream flowering-related genes like SOC1 (SUPPRESSOR OF OVEREXPRESSION OF CONSTANS 1) and FUL (FRUITFULL), and upstream GA signaling [19]; SlAHL1 and SlAHL7 in tomato show dramatic responses to ABA, salt, and drought stresses [78]; while BrAHL02 and BrAHL24 in Brassica rapa exhibit rapid up-regulation under cold and cadmium stress, peaking at 12 h [10]. Consistent with these results, our analysis revealed that BpAHL promoters are enriched in CREs associated with hormonal responses (MeJA, ABA, and SA) and environmental stresses (light, temperature, and drought) (Figure 5), suggesting conserved regulatory mechanisms across plant species.

To validate this hypothesis, we conducted transcriptomic analyses of birch subjected to various abiotic stresses (Figure 7). The results revealed distinct expression dynamics between BpAHL genes’ types: (a) Under short-term low-temperature stress, all Type-II/III BpAHLs showed significant down-regulation, either immediately or within 1.0 h (Figure 7A). (b) During prolonged high-temperature exposure, all Type-II/III members (except BpAHL05) exhibited marked suppression, reaching minimal expression at 24 h before partial recovery (Figure 7C). (c) Salt stress induced consistent gene expression patterns for BpAHL05/07/08/09/11 in both leaves and roots (Figure 7D,E), while most Type-I members (BpAHL01/06/07/12/19/21) were significantly down-regulated in roots. These type-specific and stress-dependent expression profiles suggest functional diversification of BpAHLs in environmental stress responses, with different members potentially participating in distinct regulatory pathways within the same tissue.

5. Conclusions

In summary, our study systematically identified and characterized 21 AT-hook motif nuclear localized (AHL) genes in birch (BpAHLs), all containing a PPC domain and at least one AT-hook motif. These genes are evenly spanned across birch chromosomes and primarily originated through dispersed duplication events. Phylogenetic analysis classified them into two clades (Clade-A/B) or three types (Type-I/II/III), with distinct structural features and gene expression patterns. Type-I BpAHLs (single CDSs) show root-predominant expression and evolutionary conservation, while Type-II/III members (multiple CDSs) exhibit aerial tissue-specific expression and pronounced responsiveness to diverse environmental stresses (temperature, salinity, and light), suggesting their involvement in more complex regulatory networks. These findings provide valuable genomic resources and molecular insights for birch tree improvement. Importantly, the stress-responsive BpAHLs, particularly members in Type-II/III, represent promising candidates for molecular breeding and genetic engineering applications. Their manipulation through overexpression or genome editing could potentially enhance birch adaptability to abiotic stresses, while their tissue-specific promoters may enable targeted modulation of stress tolerance mechanisms in different organs. This study thus establishes a foundation for developing stress-resilient birch varieties through biotechnological approaches.