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Genome-wide identification of five fern bHLH families and functional analysis of bHLHs in lignin biosynthesis in Alsophila spinulosa

BMC Genomics volume 26, Article number: 357 (2025) Cite this article

Abstract

Background

The basic helix-loop-helix (bHLH) transcription factors are involved in the biosynthesis of various secondary metabolites. However, genome-wide studies on the bHLH gene family in ferns and their role in lignin biosynthesis remain limited. As the second largest group of vascular plants, ferns are of significant interest for understanding plant evolution and secondary metabolism. Among ferns, Alsophila spinulosa stands out as one of the few tree ferns with a distinctive trunk structure. Investigating the genes potentially regulating lignin biosynthesis in A. spinulosa offers valuable insights into the growth and development mechanisms of its trunk, which is pivotal for the overall architecture and function of the plant.

Results

In this study, we conducted a systematic study of bHLH gene families in five ferns, including 186 in A. spinulosa, 130 in A. capillus, 107 in A. filiculoides, 71 in S. cucullata, and 67 in C. richardii. Based on phylogenetic analysis, all bHLH genes were classified into 28 subgroups. The number of bHLH members in different ferns was closely related to their growth patterns and life habits, with the number in tree ferns being much larger than in other ferns. In addition, we identified tandem duplication in C. richardii and A. capillus as a key driver of their bHLH gene diversity, whereas in A. spinulosa, segmental duplication contributed more to gene expansion and evolution. Most of the bHLH genes in ferns are in a state of purifying selection. Additionally, tissue-specific expression patterns of AspbHLH genes suggest diverse functional roles in plant growth, development, and metabolite synthesis. We further focused on three genes, AspbHLH80, AspbHLH120, and AspbHLH185, which are specifically highly expressed in xylem. Results from weighted gene co-expression network analysis (WGCNA) and downstream target gene prediction indicate their potential regulatory roles in lignin biosynthesis.

Conclusion

This study presents a comprehensive genomic analysis of the bHLH gene family in five fern species. We found a strong correlation between bHLH gene number and fern growth morphology, with tree ferns exhibiting a significantly higher number of bHLH genes. Tandem duplications were key to bHLH gene diversity in C. richardii, A. capillus, and A. spinulosa, while segmental duplications contributed more to bHLH gene expansion in A. spinulosa. Evolutionary analysis indicated most fern bHLH genes are under purifying selection. Tissue-specific expression patterns of AspbHLH genes suggest roles in growth, development, and secondary metabolism. Furthermore, WGCNA and target gene predictions highlight three genes (AspbHLH80, AspbHLH120, and AspbHLH185) potentially involved in lignin biosynthesis. Overall, this work provides key insights into the mechanisms of wood formation in ferns and advances our understanding of plant secondary metabolism.

Peer Review reports

Background

The bHLH transcription factor (TF) is a member of a superfamily widely distributed in plants, animals, and fungi [1]. bHLH proteins contain a highly conserved bHLH domain, comprising approximately 60 amino acids, which is divided into two functional regions: the basic domain and helix-loop-helix (HLH) domain. The N-terminal basic region consists of approximately 10–17 amino acids, typically including five and six basic residues, which participate in DNA binding to regulate gene expression. At the C-terminus, the HLH domain, composed of roughly 40 amino acids, forms two amphipathic α-helices connected by a loop, enabling the formation of homodimers or heterodimers to facilitate protein interactions [2, 3]. Plant bHLH TFs are typically categorized into 15–26 subfamilies based on conserved structural domains, phylogenetic relationships, and sequence homology [4]. When atypical bHLH proteins are included, the classification can extend to 35 subfamilies [5]. The identification of the bHLH gene family in plants dates back to 1989 [6]. Since then, advancements in genome sequencing technologies have enabled extensive identification and investigation of the bHLH superfamily across a diverse range of plant species, including Prunus mume [3], Litsea cubeba [7], Cyclocarya paliurus [8], Malus domestica [9], and others. Conducting a genome-wide characterization of bHLH TF is essential for advancing our understanding of the transcriptional mechanisms and functional roles attributed to the bHLH gene family. Numerous studies have highlighted the functional diversity of bHLH proteins, demonstrating their involvement in various biological processes. These include anthocyanin biosynthesis [10], the regulation of plant secondary metabolism [11], plant growth and development [12], and responses to stress [13, 14].

Pteridophytes are the second largest vascular plants on earth next to angiosperms. Vascular tissues in plants are crucial for providing physical support and transporting water, sugars, hormones, and other small signaling molecules throughout the plant [15]. The vascular system consists of two main tissue types, xylem and phloem, organized into vascular bundles [16]. Ferns possess tracheid-based xylem, which contains large amounts of lignin [17, 18]. The bHLH family plays a crucial role in lignin biosynthesis. In cotton (Gossypium hirsutum), GhbHLH18 negatively regulates fiber strength and length by enhancing lignin biosynthesis in fiber [19]. In Populus trichocarpa, overexpression of PtrbHLH186 caused abnormal lignification and enhanced vessel cell development [20]. In Arabidopsis thaliana, the heterologous expression of the sorghum transcription factor SbbHLH1 downregulates lignin biosynthesis [21]. Although bHLH TF families have been identified in many species, they have not yet been reported in ferns. To date, five fern genomes have been published, including A. spinulosa [22], A. filiculoides [23], S. cucullata [23], C. richardii [24], and A. capillus [25], paving the way for bHLH family investigations in ferns. These species represent different types of ferns, including aquatic ferns, terrestrial ferns, and tree ferns, spanning different evolutionary levels [22,23,24,25]. This selection provides a unique opportunity to study the evolution of gene families across distinct fern lineages, revealing how gene functions may diverge under varying environmental pressures and ecological niches. Among the five ferns, A. spinulosa is a tree fern with a large, erect rhizome compared to the other species, making it an ideal model for studying xylem development. In a previous study [22], we identified many G-type lignins in A. spinulosa xylem, but the transcriptional regulatory mechanisms of lignin biosynthesis remain unclear. Therefore, identifying bHLH genes in five ferns and determining the functional roles of A. spinulosa bHLHs in lignin biosynthesis are of great significance.

Here, we report a genome-wide analysis of the bHLH family in five ferns, identifying a non-redundant set of 558 bHLH genes in total. These bHLH proteins were comprehensively and systematically analyzed, including categorization, physicochemical properties, phylogenetic relationships, chromosome distribution, conserved motifs, and protein interactions. Expression patterns of AspbHLH gene in stem (xylem, pith, phloem, sclerenchymatic belt) and leaves were analyzed using transcriptome data and Quantitative Real-time PCR (qRT-PCR). Three candidate AspbHLHs (AspbHLH80, AspbHLH120, AspbHLH185) potentially involved in lignin biosynthesis were identified. This study provides not only the evolutionary relationship of the bHLH family in ferns but also a theoretical basis for understanding the regulatory mechanisms of AspbHLH TFs in lignin biosynthesis.

Materials and methods

Plant materials

The A. spinulosa materials used in this study were sampled from MeiShan City, Sichuan Province, China (29.90°N, 103.14°E). Tissues, including xylem (Xy), phloem (Ph), pith (Pi), sclerenchymatic belt (Sb), and leaf (Le), were collected from three individual A. spinulosa trees as biological replicates. The samples were immediately frozen in liquid nitrogen and stored at -80℃ for RNA extraction.

Identification and classification of bHLH genes in five ferns

The genome sequence, protein sequences, and annotation file of A. spinulosa and A. capillus were obtained from the FigShare database (https://doiorg.publicaciones.saludcastillayleon.es/10.6084/m9.figshare.19075346; BioProject number: PRJCA006485) (https://figshare.com/s/47be9fe90124b22d3c0e; BioProject number: PRJNA593372) [22, 25]. The genome data of A. filiculoides and S. cucullata were obtained from the FernBase database (https://www.fernbase.org; BioProject number: PRJNA430527 and PRJNA430459.) [23]. The genome data for C. richardii were obtained from the Phytozome database (https://phytozome-next.jgi.doe.gov/info/Crichardii_v2_1; BioProject number: PRJNA729743) [24]. A comprehensive hidden Markov model (HMM) of the bHLH domain (PF00010) was obtained from the Pfam database (https://pfam.xfam. org). The bHLH genes were identified using HMMER 3.0 with default settings and and an E-value threshold of 1e− 10 [26]. The structural details of each bHLH gene identified in the five ferns were further analyzed using SMART and NCBI-CDD online tools to confirm the presence of the bHLH domain. The ExPASy server (http://web.expasy.org/protparam/) was used to calculate hydropathicity (GRAVY), isoelectric point (PI), and molecular weight (MW) [27]. Subcellular localization (SL) was predicted using WoLF PSORT online software (https://www.genscript.com/wolf-psort.html).

Phylogenetic analysis and classification of bHLH genes

To understand evolutionary relationships and classify bHLH genes, a rooted neighbor-joining (NJ) phylogenetic tree of five ferns and Arabidopsis bHLH proteins was constructed using the MEGA 7 program [28]. The TAIR11 database (https://www.arabidopsis.org) provides sequences of Arabidopsis bHLH proteins. All protein sequences were aligned using the default settings of ClustalW. The phylogenetic tree was visualized and enhanced using Evolview2 [29].

Analysis of gene architecture and conserved motifs

The bHLH protein sequences from the five ferns were analyzed using the MEME online program (http://meme-suite.org/) to predict conserved motifs [30]. The discovery number of the pattern was set to 10, with the minimum and maximum motif widths and repetition parameters specified. The bHLH motif domains were visualized using TBtools [31]. The online Gene Structure Display Server (GSDS) (http://gsds.cbi.pku.edu.cn/index.php) was used to show the gene structure and extract the locations of every bHLH gene sequence from the genome annotation file.

Analysis of chromosomal locations, gene duplication and collinearity

The A. spinulosa, A. capillus, and C. richardii bHLH genes were mapped to their respective chromosomes. Chromosomal localization analysis was not performed for A. filiculoides and S. cucullata due to the lack of chromosome-level genome assemblies. Map Chart software was used to visualize their distribution [32]. Software MCScanX was used to analyze gene duplication [33]. All protein sequences of A. spinulosa were compared against each other using BLASTP [34]. By using the KaKs­Calculator, the values of non-synonymous (Ka) and synonymous (Ks) words were calculated [35]. MCScanX was used to identify the synteny relationship of homologous bHLH genes obtained from A. spinulosa and other ferns (A. capillus, A. filiculoides, S. cucullata, and C. richardii).

Promoter cis-regulatory element analysis of bHLH Genes

The 2000 bp upstream sequence of the bHLH gene was extracted from the genome file and uploaded to the Plant-Care website (http://bioinformatics.psb.ugent.be/webtools/plantcare/html) for the prediction of cis-acting elements. The results of the Plant-Care study were condensed and TBtools was utilized to visualize them [31].

Expression pattern analysis of AspbHLH genes

To further explore the expression patterns of the AspbHLH gene family in different tissues and organs, transcriptomic data for five different tissues were obtained from our previous studies [22], which are stored in the public database (BioProject: PRJCA006485). For xylem (Xy), phloem (Ph), sclerenchymatic belt (Sb) in the vascular bundle, pith (Pi) and leaf (Le), mature stems were harvested, and transverse sections were cut. The individual tissues were carefully separated using a scalpel, with xylem, phloem, and sclerenchymatic belt tissues dissected from the vascular bundle, and the pith tissue obtained from the central portion of the stem. All tissues were collected from three individual trees as biological replicates. Data processing and visualization were performed using TBtools software [31].

qRT-PCR analysis

Total RNA was extracted from xylem (Xy), phloem (Ph), and sclerenchymatic belt (Sb) in the vascular bundle, pith (Pi), and leaf (Le) sample using the CTAB method [36]. The qRT-PCR was performed with reference to previous studies, with AspiActin selected as an internal control, and the primers are shown in the Supplementary Table 1 [22]. In this study, the relative expression levels of genes in various samples were computed using the 2−ΔΔCt method. Excel 2019 software was used to align and compute all the data. Using the SPSS program, an analysis of variance (ANOVA) was carried out with p < 0.05 as the significance threshold. GraphPad Prism 5 was used to create the graphs [37].

WGCNA analysis and functional annotation

To further investigate the potential roles of AspbHLH genes in different tissues, we obtained transcriptome data from 10 tissues (leaves and stems at three different developmental periods, and xylem, phloem, pith, and sclerenchymatic belt) of A. spinulosa, totaling 30 samples from a previous study [22], which were subsequently used for WGCNA. The analysis was performed using the WGCNA (v1.69) package in R (v3.6.1) [38]. Using a topology-overlap-based dissimilarity metric, all genes were subjected to hierarchical clustering, and a gene dendrogram was constructed based on the Topological Overlap Matrix (TOM). Gene expression profiles for each module identified through the gene dendrogram were computed to assess associations with various plant tissues. Within the co-expression network, the edge weights between any two connected genes were determined based on their topological overlap measure, ranging from 0 to 1. Visualization of the network was performed using Cytoscape (v.3.8.2) software. Functional annotations of genes within modules, including Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway enrichment analyses, were conducted following established methodologies from prior studies [22].

Subcellular location analyses of AspbHLH genes

To further investigate the subcellular localization of AspbHLH80, AspbHLH120, and AspbHLH185 genes, we amplified the stop codon-free coding sequences (CDS) and inserted them into the pCAMBIA1300 vector under the control of the CaMV 35 S promoter, with green fluorescent protein (GFP) tagging. The amplification primers for plasmid construction were designed using SnapGene 6.0.2 software (Supplementary Table 2). Subsequently, the constructed plasmid was introduced into Agrobacterium GV3101 using the conventional freeze-thaw method. The experimental setup involved the nuclear localization vector NLS-RFP and the empty vector pCAMBIA1300-GFP as the control group. The experimental group consisted of the recombinant plasmid pCAMBIA1300-AspbHLH-GFP and the nuclear localization vector NLS-RFP. These constructs, both experimental and control, were infiltrated into 5-week-old tobacco leaves using an expression buffer (10 mM MES pH 5.6, 10 mM MgCl2, 200 µM acetosyringone) [39]. The infiltrated tobacco was then kept in the dark for 24 h, followed by a 48-hour incubation under low light conditions. Subsequently, the GFP signal was detected using Leica confocal microscopy.

Transcription factor binding site analysis of AspbHLH80, AspbHLH120, and AspbHLH185

To further elucidate the transcription factor (TF) binding sites of AspbHLH80, AspbHLH120, and AspbHLH185 within the AspbHLHs family, target genes were scanned using the Binding Site Prediction Tool available on the Plant Transcriptional Regulatory Maps website (http://plantregmap.gao-lab.org/).

Results

Identification and classification of bHLH genes in five ferns

After removing redundant and structurally incomplete candidate genes, a total of 561 bHLH genes were identified. Specifically, for the species A. spinulosa, A. capillus, A. filiculoides, S. cucullata, and C. richardii, 186, 130, 107, 71, and 67 bHLH genes were respectively identified, respectively (Table 1). Compared to the other four species, A. spinulosa exhibits the highest number of bHLH genes (186), and the proportion is the highest. The number of bHLH genes in C. richardii and A. capillus are similar, while A. filiculoides and S. cucullata have comparable numbers of bHLH genes. Based on the chromosomal locations of the genes, bHLH genes from the five species were named. The average molecular weight and isoelectric point of these proteins ranged from 59.16 to 45.01 and 6.47 to 6.96, respectively. Subcellular localization predictions suggest that most of these proteins function in the cell nucleus, with a smaller fraction predicted to be located in other cellular compartments such as chloroplasts. Additional details and physical properties can be found in Supplementary Table 3.

Table 1 Number of bHLH genes in five ferns species
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Phylogenetic analysis reveals evolutionary divergence and subfamily distribution of bHLH proteins in five fern species

To further investigate the evolutionary relationships between the 173 bHLH family members in A. thaliana and the 561 bHLH proteins in five ferns, a rooted phylogenetic tree was constructed using the neighbor-joining method (Fig. 1). Arabidopsis was used as an outgroup. The 561 bHLH gene family members were classified into 30 subfamilies, with detailed information available in Supplementary Table 4. It is worth noting that none of the five ferns exhibited clustering of bHLH genes in subfamilies B17(X), B19(XV), and 30. Notably, subfamily B15(III(a + c)) stands out with the highest number of bHLH genes (60), while subfamily B23(VIIIa) is characterized by the lowest number of bHLH genes (1). Within the same subfamily, there is a substantial difference in the number of bHLH genes among the five ferns. For instance, in subfamily XIV, A. spinulosa possesses 21 members, while C. richardii and A. capillus have 5 members each, and S. cucullata and A. filiculoides exhibit only 3 and 1 member(s), respectively. Similar variations are observed in other subfamilies. The above results indicate that bHLH family proteins in the five ferns exhibit significant diversity and differentiation throughout the evolutionary process, providing valuable insights for future studies aiming at uncover their specific biological functions.

Fig. 1
figure 1

Systematic phylogenetic analysis of bHLH proteins in five ferns and A. thaliana. Different colors represent different groups, and all bHLH proteins are clustered into subclades based on the priority classification rule of A. thaliana bHLH proteins. Different shapes along the phylogenetic tree represent bHLH proteins from different species

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Gene structure and protein motif diversity among bHLH genes in five ferns

The results showed that the structures of the 561 bHLH genes differ significantly (Supplementary Fig. 1A-E). Some of the genes possess multiple UTR regions, while a few lack this structure. This structural diversity may reflect evolutionary differences between homologous bHLH genes. In addition, bHLH genes clustered in the same group tend to have similar exon/intron structures, exon numbers, and gene lengths. The distribution of bHLH protein motifs in the same subgroups was also broadly similar, suggesting that motif distribution may be functionally related. The motifs of bHLH proteins in five ferns were analyzed using the MEME online software tool, and the 20 motifs obtained were compared and named motifs 1 to 20 (Supplementary Table 5). Motif 1 is the basic region and the first helix, and motif 2 contains the second helix, both of which are bHLH-specific structural domains, and both motifs are identified in all bHLH proteins.

Cis-regulatory element diversity in the promoter regions of bHLH genes in five fern species

Cis-regulatory elements (CREs) in promoter regions are typically located upstream of the 5’ end of genes, where they bind to transcription factors (TFs) and play a crucial role in regulating gene expression. In this study, we predicted 561 CREs in the promoter region of the bHLH gene. Based on their function of cis-regulatory elements and response to the environment, we classified all CREs into 15 categories. Further details on the cis-regulatory elements of the five ferns can be found in Supplementary Tables 6 − 1. The results showed that light-responsive elements accounted for most of the regulatory elements identified, followed by phytohormone-responsive and abiotic stress-responsive elements (Supplementary Tables 6 − 2). In addition, the number of MeJA and ABA response elements was much higher than that of other phytohormone response elements (Supplementary Fig. 2A-D). Subfamily members such as B5(Ib(1)), B14(III(d + e)), and XIV possessed a large number of MeJA and ABA response elements in A. spinulosa (Fig. 2A). The results also revealed that 13 AspbHLH family members had five phytohormone-responsive cis-regulatory progenitors in their promoter sequences (Fig. 2B). These results suggest that phytohormones, particularly MeJA and ABA, play important roles in regulating the expression of bHLH genes in the five fern species.

Fig. 2
figure 2

The cis-regulatory elements analysis of bHLH promoter regions in A. spinulosa. Promoter cis-acting elements in A. spinulosa. Different colors represent different types of cis-acting elements. (A) The number of CREs identified in the putative promoters of AspbHLHs. The bar chart shows the frequency of each type of CRE in the promoters of all AspbHLHs; (B) Venn diagram of various CREs. Venn diagram illustrating the overlap and distribution of different CREs across the promoter regions of AspbHLHs. This diagram highlights shared and unique cis-regulatory elements across the promoters

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Diverse chromosomal distribution and evolutionary divergence of bHLH genes across three fern species

Since the genomes of A. filiculoides and S. cucullata have not been assembled to the chromosome level, chromosomal location information of bHLH genes was only available for the remaining three ferns species. The results showed considerable variation in the distribution of bHLH genes on chromosomes. A. spinulosa has 69 chromosomes, with the AspbHLH genes distributed across only 28 of these chromosomes (Fig. 3A). Chromosome 2 has the most AspbHLH members, while chromosomes 12, 24, 25, and 26 have no AspbHLH genes. Each of the remaining chromosomes contains at least one AspbHLH gene. A. capillus has 30 chromosomes, with AdcbHLH genes distributed on 29 of them; only chromosome 20 lacks of AdcbHLH genes. A. capillus has 39 chromosomes, and the AdcbHLH genes are distributed across 34 of these, with chromosome 20 having the most AspbHLH members.

Fig. 3
figure 3

Chromosomal locations and Segmental duplication of bHLH genes. (A) A. spinulosa. (B) A. capillus. (C) C. richardii. The red arcs indicate regions of tandem duplication, while the yellow squares highlight the specific chromosomal regions where tandem duplications occur. The numbers represent the positions of the bHLH genes on the respective chromosomes (M), illustrating the distribution of bHLH genes and their duplication events

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Tandem duplications of bHLH genes in A. spinulosa (11), C. richardii (19), and A. capillus (11) were identified using MCScanX (Fig. 3A-C), and several tandemly duplicated bHLH genes were found on the chromosomes of the three ferns. By calculating the Ka/Ks ratios of tandemly duplicated gene pairs, we gained insights into the evolutionary constraints on the bHLH gene family members. It was found that AspbHLH19 & AspbHLH20 and CribHLH45 & CribHLH46 have Ka/Ks values greater than 1, indicating that they evolved under positive selection pressure (Supplementary Table 7). The remaining tandemly duplicated genes have Ka/Ks values below 1, indicating that they have undergone purifying selection during their evolution. In contrast, tandem duplicate genes with Ka/Ks ratios close to 1 have undergone neutral evolution.

Gene duplication and synteny analysis of AspbHLHs

To further investigate the amplification mechanism of the AspbHLH gene family, a total of 76 gene duplication events were identified in this study (Fig. 4A), leading to the formation of gene pairs within AspbHLHs. Gene duplication events were not detected in C. richardii and A. capillus (Supplementary Fig. 3A-B). To explore the evolutionary relationship of bHLH genes among different ferns, we subjected two terrestrial ferns, C. richardii and A. capillus, and two aquatic ferns, A. filiculoides and S. cucullata with A. spinulosa, to covariance analysis. It was found that the number of covariate genes was closely related to the living environment of different ferns. Specifically, 53 covariant genes were found in A. spinulosa and A. capillus, while 4, 7, and 7 covariant genes were found in C. richardii, A. filiculoides, and S. cucullata, respectively (Supplementary Fig. 3C-F, Supplementary Table 8). This suggests that ferns from similar habitats share more covariant genes. In addition, the number of bHLH gene covariates differed significantly between aquatic and terrestrial ferns (Fig. 4B). C. richardii exhibited less covariance with A. filiculoides, possibly due to the more distant evolutionary relationship between these two species. We also observed that some genes have multiple homologs in the other two species. For example, the gene in Chr01 of A. spinulosa had two collinearity genes in Chr17 and Chr25 of A. capillus. The above results suggest that the fern bHLH gene family may have expanded during evolution.

Fig. 4
figure 4

The Segmental duplication in A. spinulosa and collinearity analysis of bHLH genes in different species. (A) Red lines connect fragments of duplicated gene pairs, while grey lines represent co-linear blocks within A. spinulosa. (B) Multi-collinearity analysis of bHLH genes across five fern species. A blue line between two species indicates collinearity of genes

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Expression patterns of AspbHLH genes across five tissues

To deeply uncover the role of bHLH family genes and their expression patterns in the developmental process, the expression of AspbHLH gene was extracted from the transcriptome data in five tissues: xylem (Xy), phloem (Ph), sclerenchymatic belt (Sb) in the vascular bundle, pith (Pi) and leaf (Le) expression in five tissues (Supplementary Table 9). A circular heatmap (Fig. 5A) was created, with a cross-sectional view of the A. spinulosa stem at the center of the heatmap. The locations of the different tissues are marked with arrows in the figure. The results showed that AspbHLH genes were expressed in five different groups, but some genes showed tissue-specific expression patterns. For instance, 26 genes, including AspbHLH65, AspbHLH50, AspbHLH79, etc., were highly expressed only in leaves (Fig. 5A). AspbHLH12, AspbHLH28, AspbHLH44, AspbHLH86, and AspbHLH160 were highly expressed only in the sclerenchymatic belt. It is noteworthy that more than one-third of AspbHLHs genes were highly expressed in xylem, especially AspbHLH80, AspbHLH120, and AspbHLH185 were significantly higher than the other four tissues (Supplementary Table 10). Prediction of target genes downstream of these three genes revealed that the target genes contained many transcription factors that regulate lignin synthesis, such as NAC and MYB (Supplementary Fig. 4A-C), suggesting that AspbHLH80, AspbHLH120 and AspbHLH185 may play roles in the transcriptional regulation of xylem development and lignin synthesis. Although most AspbHLHs showed differential expression in different tissues, indicating their functional diversity in different tissues, these inferences require further validation. To validate the reliability of the transcriptome data, a random selection of 15 AspbHLH genes was subjected to further analysis of their expression patterns using qRT-PCR. The results demonstrated consistent expression patterns of these 15 genes across five different tissues, consistent with the RNA-Seq (Fig. 5B-P).

Fig. 5
figure 5

Expression patterns of bHLH genes in different organs based on transcriptome datasets. (A) Expression heatmap of AspbHLHs in xylem (Xy), phloem (Ph), sclerenchymatic belt (Sb) in the vascular bundle, pith (Pi) and leaf (Le). The center of the heat map is a cross-section of the A. spinulosa stem, with red arrows marking the locations of the different tissues. Genes that were not expressed in any tissue were excluded from the figures. The color bars represent the relative expression levels after column scale, where red indicates high expression levels and blue represents low expression levels. The data in the heatmap were the original FPKM values of the samples. (B-P) Analysis of AspbHLHs transcript levels in xylem (Xy), phloem (Ph), sclerenchymatic belt (Sb) in the vascular bundle, pith (Pi) and leaf (Le). Blue line represents the transcript levels of AspbHLHs determined by RT-qPCR, and red bar graph indicates the FPKM value. Values are from three technical replicates and three biological replicates, with error bars indicating standard deviations

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Identification of key AspbHLH genes involved in xylem development and lignin biosynthesis

Previous results indicated that many AspbHLH genes were highly expressed in xylem. To further understand the potential functions of AspbHLH genes during xylem development or lignin synthesis, we performed WGCNA analysis using RNA-Seq data from five tissues of A. spinulosa, yielding a total of 14 clustered dendrogram modules (Fig. 6A). Among them, the network in the yellow module had a strong correlation with xylem (Fig. 6B). GO and KEGG enrichment analysis of the genes in the yellow module revealed that many of them were enriched in the secondary metabolite synthesis pathway and the phenylpropanoid biosynthesis pathway, among others (Fig. 6C-D, Supplementary Tables 11–12). Notably, some of the genes were significantly enriched in xylem development and lignin biosynthesis. In-depth analysis revealed that AspbHLH80, AspbHLH120, and AspbHLH185 were also located in the yellow module, and they were potentially associated with several key enzyme genes related to lignin synthesis, such as AspiHCT45, Aspi4CL5, and AspiCOMT2(Fig. 6E), which were also highly expressed in xylem (Fig. 6F). These findings further support the potential role of the AspbHLH genes in lignin biosynthesis and xylem development.

Fig. 6
figure 6

Results of WGCNA analysis and KEGG, GO analysis of yellow module. (A) Hierarchical clustering of gene modules in 10 tissues of A. spinulosa, including leaves, stems at three different developmental periods, xylem, phloem, pith, and sclerenchymatic belt. (B) Heatmap showing the gene co-expression network across different modules in different tissues. (C) GO analysis for the yellow module. (D) KEGG analysis for the yellow module. (E) Co-expression network of three differentially expressed bHLH genes (AspbHLH80, AspbHLH120, and AspbHLH185) in xylem, co-expressed with genes related to lignin biosynthesis. Different colors represent the number of co-expressed genes, with redder colors indicating a higher number of co-expressed genes. Genes highlighted in red indicate co-expression with the three bHLH genes. (F) Expression patterns of lignin biosynthesis-related genes co-expressed with AspbHLH80, AspbHLH120, and AspbHLH185 in various tissues. The heatmap data represents the original FPKM values of the samples

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Subcellular localization of AspbHLHs

Normally, transcription factors (TFs) play a role in transcriptional regulation in the nucleus [40]. To further explore the subcellular localization of the highly expressed AspbHLH80, AspbHLH120, and AspbHLH185 genes involved in transcriptional regulation in the xylem, we predicted their subcellular localization using the WoLFPSORT online tool. The results indicated that all three genes were located in the nucleus. To validate these predictions and further investigate their potential role in regulating gene expression, we transiently expressed 35 S: GFP-AspbHLH80, 35 S: GFP-AspbHLH120, and 35 S: GFP-AspbHLH185 fusion proteins in tobacco. Confocal microscopy analysis confirmed that these three genes were indeed localized in the nucleus (Fig. 7), suggesting that AspbHLH80, AspbHLH120, and AspbHLH185 might be involved in regulating gene expression.

Fig. 7
figure 7

Subcellular localization of AspbHLH80, 120 and 185 proteins in N. benthamiana leaves

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Discussion

A. spinulosa is the only extant woody fern that distinguishes itself from other ferns by the presence of vascular tissue in its main stem, which provides a strong structural support [22]. The emergence of this organization is an important symbol of the evolution of aquatic plants to land plants [41]. However, lignin is an important component of the secondary cell wall of the xylem in vascular tissues, which provides mechanical support in fiber cells and aids in water transport [42]. Studying lignin synthesis and its transcriptional regulation in Cyathea is crucial for understanding trunk formation and development during the evolutionary shift from aquatic to terrestrial ferns. The bHLH family of transcription factors, the second-largest family of transcription factors in plants, is involved in regulating various metabolic processes [43]. Previous study have shown that bHLH transcription factors are associated with abiotic stress and the synthesis of secondary metabolites in species such as L. cubeba, C. paliurus, and Artemisia argyi [7–8, 44], ith evidence suggesting their potential involvement in lignin biosynthesis [20]. Despite limited research on the bHLH gene family in ferns, recent genomic studies of ferns provide an opportunity for a comprehensive analysis and characterization of the bHLH family at the genomic level [22,23,24,25].

In this study, we observe a significant correlation between the number of bHLH family genes identified in the five ferns and their respective habitats. Terrestrial ferns exhibited a higher abundance of bHLH genes compared to aquatic ferns, with this pattern being particularly evident in A. spinulosa(Fig. 8). This suggests that the expansion of the bHLH gene family may play a key role in fern growth and development and adaptation to complex environments [45]. Terrestrial ferns are exposed to more variable climatic and soil conditions and have more complex root systems and above-ground structures [46], which may require additional genes to cope with these challenges. This mechanism is prevalent in the plant kingdom; for example, a significant expansion of WRKY transcription factors has helped Primulina huaijiensis adapt with high salinity and water scarcity [47]. In addition, the significant expansion of HAT and LIMYB gene family members in Tamarix chinensis increased its disease resistance [48]. Our phylogenetic analysis suggests that bHLH gene diversification in ferns is shaped by ecological adaptation, with expansions in tree ferns likely linked to structural and metabolic needs. Promoter cis-acting elements regulate gene expression, and typically, the greater the number of elements, the higher the level of transcription [49, 50]. Insights into promoter structures can provide clues about possible functions and regulatory mechanisms [51]. Research has found that the light responsive elements in the promoter region of the bHLH gene are the most abundant regulatory elements. The second most abundant responsive element is the hormone-related element, with the ABA responsive element having the highest number. ABA is closely related to stress and can increase the plant’s resistance to stress [52], indicating that some bHLH genes may be induced by ABA to affect the development or stress of ferns. These findings align with research on Prunus sibirica, which also suggests ABA’s role in regulating stress responses [53].

Fig. 8
figure 8

Model of bHLH gene family evolution and regulation in ferns: insights into lignin biosynthesis. Xy: xylem, Ph: phloem, Sb: sclerenchymatic belt, Pi: pith. The numbers on the species indicate the number of bHLH family members, and the dashed lines represent potential regulatory relationships

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Genomic evolution of the bHLH gene in A. spinulosa is largely driven by gene duplication events, which include tandem and segmental duplications [54, 55]. Through gene duplication, plants can acquire new functions and expand their adaptations [56]. MCScanX analysis revealed the identification of many tandemly duplicated genes in the fern genome, most of which were found under purifying selection (Supplementary Table 5), although some genes are under positive selection. Based on these findings, we hypothesize that gene duplication events are the main factors driving the evolution of fern bHLH genes [57]. In addition to segmental and tandem duplications, gene families also undergo gene loss events during evolution, which result in subgroups with varying numbers of genes per subfamily across different species [58]. Phylogenetic tree analysis showed that the 561 bHLH genes could be classified into 28 subfamilies, which is consistent with the results of Carthamus tinctorius [59]. The absence of bHLH genes in three subfamilies (X, XV, and 30) compared to Arabidopsis may be attributed to gene loss or natural selection, which led to the elimination of these genes during fern evolution.

Gene expression patterns reflect their functional roles to some extent [37]. A. spinulosa bHLH family members exhibit differential expression across various tissues (Fig. 5). This study identified certain AspbHLH genes that display tissue-specific expression patterns, such as AspbHLH50 and AspbHLH65, which are predominantly expressed in leaves. These genes may be associated with leaf development and stress resistance [60]. Genes such as AspbHLH70 and AspbHLH136 exhibit high expression in the pith. Previous research has found that sassafras pith is rich in various secondary metabolites such as starch, phenols, and terpenoids [22]. Moreover, substantial evidence suggests a correlation between bHLH genes and the synthesis of secondary metabolites, including terpenoids and alkaloids [44, 61]. Therefore, it is speculated that these bHLH genes highly expressed in the pith may be associated with the synthesis of secondary metabolites. Notably, AspbHLH80, 120, and 185 are exclusively highly expressed in the xylem, with no detection in other tissues (Fig. 8). Although bHLH genes are not direct regulatory factors of lignin biosynthetic enzymes, studies have demonstrated their ability to indirectly regulate lignin biosynthesis by binding to other transcription factors. For example, in G. hirsutum, GhbHLH18 binds to the E-box in the GhPER8 promoter, regulating its expression, and GhPER8 subsequently catalyzes lignin biosynthesis, which is crucial for fiber strength and elongation [19]. Overexpression of PtrbHLH186 in P. trichocarpa resulted in retarded growth, increased guaiacyl lignin, a higher proportion of smaller stem vessels, and enhanced drought tolerance, with PtrMYB074 and PtrWRKY19 transactivating PtrbHLH186 through dimer formation [20]. A similar study found that the Sorghum transcription factor SbbHLH1, when heterologously expressed in Arabidopsis, downregulated lignin biosynthesis [21]. Overexpression of PagUNE12 in P. alba resulted in significantly reduced plant height, shorter internodes, and curled leaves. Additionally, it promoted the development of secondary xylem, leading to thicker secondary cell walls compared to wild-type P. alba [62]. Based on these findings, we speculate that the three genes may be involved in the transcriptional regulation of lignin biosynthesis, but their specific functions require further validation.

WGCNA can cluster genes with similar expression patterns and identify interactions among these genes [38]. These genes may exhibit similar functions or participate in the same signaling pathways or physiological processes [63]. In this study, WGCNA analysis focused on studying a key module associated with wood. Enrichment analysis revealed that genes in the yellow module are enriched in various pathways, including the lignin biosynthesis pathway. Notably, the wood-specifically expressed genes AspbHLH80, AspbHLH120, and AspbHLH185 are also found within this module. Further analysis showed that these three genes exhibit co-expression relationships with other genes related to wood formation. Additionally, downstream target gene prediction suggests their potential interaction with key transcription factors regulating lignin biosynthesis, such as NAC and MYB [64]. Therefore, we speculate that these three genes may be involved in regulating lignin synthesis. However, the exact mechanism by which AspbHLH80, AspbHLH120, and AspbHLH185 regulate lignin synthesis remains to be further investigated. Future studies should focus on functional analysis through overexpression or silencing of these genes in A. spinulosa, coupled with metabolite profiling of lignin biosynthesis intermediates. Additionally, identifying their target genes using chromatin immunoprecipitation (ChIP) assays and exploring their interactions with key enzymes in the lignin biosynthetic pathway will provide a deeper insight into their regulatory roles.

The findings of this study hold significant potential for advancing plant biotechnology and genetic improvement, particularly in agricultural and industrial applications. The bHLH genes identified in this research are likely involved in key processes like growth regulation and lignin biosynthesis, which are crucial for enhancing plant productivity and biomass quality. For example, manipulating genes involved in lignin biosynthesis can improve the quality of biomass for bioenergy production by optimizing the lignin content for better degradation or enhancing lignin deposition for structural strength [65]. These strategies can be particularly valuable in industries such as biofuels and paper production [66]. Additionally, understanding the function of bHLH genes in lignin biosynthesis offers opportunities to improve wood quality in forestry. By controlling the expression of bHLH genes, it may be possible to create plants with enhanced or reduced lignin content, optimizing wood for various industrial applications [21]. This has broad implications, not only in agriculture but also in the development of sustainable materials and energy sources [67].

Conclusion

In this study, we systematically investigated the bHLH gene families of five fern species at the genomic level and conducted a series of analyses and validations. We identified 561 members of the bHLH gene family, which were classified into 28 subfamilies. Our results revealed a close correlation between the number of bHLH members in ferns and their growth morphology and lifestyle, with tree ferns exhibiting a significantly higher number of bHLH members compared to other ferns. Tandem duplication was found to be a key driver of bHLH gene diversity in C. richardii, A. capillus, and A. spinulosa, while segmental duplication contributed more significantly to the expansion and evolution of bHLH genes in A. spinulosa. During evolution, most bHLH genes in ferns were under purifying selection. Additionally, tissue-specific expression patterns of AspbHLH genes were observed, indicating their diverse roles in plant growth, development, and metabolite synthesis. We also highlighted the potential functions of three genes specifically expressed in the wood, with WGCNA and downstream target gene prediction analyses suggesting their potential regulatory roles in lignin synthesis. In conclusion, this research provides valuable insights into the mechanisms and regulatory networks underlying wood formation in ferns and contributes to our understanding of plant secondary metabolism.

Data availability

The genome sequence, protein sequences, and annotation file of A. spinulosa and A. capillus were obtained from the FigShare database (https://doiorg.publicaciones.saludcastillayleon.es/10.6084/m9.figshare.19075346; BioProject number: PRJCA006485) (https://figshare.com/s/47be9fe90124b22d3c0e; BioProject number: PRJNA593372). The genome data of A. filiculoides and S. cucullata were obtained from the FernBase database (https://www.fernbase.org; BioProject number: PRJNA430527 and PRJNA430459.). The genome data for C. richardii were obtained from the Phytozome database (https://phytozome-next.jgi.doe.gov/info/Crichardii_v2_1; BioProject number: PRJNA729743). The transcriptome data of 10 tissues (leaves and stems at three different developmental periods, and xylem, phloem, pith, and sclerenchymatic belt) of A. spinulosa, were retrieved from the National Center for Bioinformation database under the BioProject no. PRJCA006485.

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Acknowledgements

The author thanks the lab members for assistance. This study was carried out with the support of molecular laboratory of college of forestry, sichuan agricultural university.

Funding

This research was funded by Sichuan Province Innovative Talent Funding Project for Postdoctoral Fellows (X H, 2322999070), National Natural Science Foundation of China (X H, 32301612) and Natural Science Foundation of Sichuan Province (X C, 2022NSFSC0135; X H, 2025ZNSFSC0994) and China Postdoctoral Science Foundation (X H, 2023M742510). The funding bodies had no role in the experimental design, sample collection, data analysis and interpretation, and manuscript writing.

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Author notes

  1. Xiong Huang and Jiangtao Fan contributed equally to this work.

Authors and Affiliations

  1. Key Laboratory of Ecological Forestry Engineering of Sichuan Province, College of Forestry, Sichuan Agricultural University, Chengdu, 611130, China

    Xiong Huang, Jiangtao Fan, Hongfei Li, Gang Wang & Xiaohong Chen

  2. National Forestry and Grassland Southwest Engineering Technology Research Centre of Taxus, Sichuan Agricultural University, Dujiangyan, 611800, China

    Xiong Huang, Jiangtao Fan & Gang Wang

  3. Sichuan Forestry and Grassland Science and Technology Extension Station, Chengdu, 610081, China

    Cai Liu

  4. Sichuan Provincial Forestry Station General, Chengdu, 610081, China

    Peiyun Wang

Authors
  1. Xiong Huang
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  2. Jiangtao Fan
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  3. Cai Liu
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  6. Gang Wang
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  7. Xiaohong Chen
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Contributions

XC and GW formulated and designed the experiments; CL and PW collected the materials; JF and XH performed the experiments; JF and HL drawn the figures, analyzed the data and wrote the paper; XC and GW revised and proofread the paper. All authors read and approved the final manuscript.

Corresponding authors

Correspondence to Gang Wang or Xiaohong Chen.

Ethics declarations

Ethics approval and consent to participate

The A. spinulosa materials were obtained from MeiShan City, Sichuan Province, Professor Xiong Huang ensures that we have permission to collect and culture the A. spinulosa. Meanwhile, Professor Xiaohong Chen, undertook the formal identification of the plant material used in the present study. The study was conducted in accordance with the relevant guidelines in Sichuan Agricultural University.

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Not applicable.

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The authors declare no competing interests.

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Supplementary Material 1

: Supplementary Fig.1. Evolutionary tree, motifs and gene structures of the five ferns bHLH. Different colors represent different motifs, and gene structures. (A) A. spinulosa. (B) A. filiculoides, (C) S. cucullata (D) A. capillus. (E) C. richardii.

Supplementary Material 2

: Supplementary Fig.2. The cis-regulatory elements analysis of bHLH promoter regions in four ferns. (A) A. capillus, (B) A. filiculoides, (C) C. richardii. (D) S. cucullata.

Supplementary Material 3

: Supplementary Fig.3. The synteny analysis of bHLH genes between A. spinulosa and other four ferns and Segmental duplication of A. capillus and C. richardii bHLH genes. (A-B) the grey lines are co-linear blocks in the A. capillus and C. richardii. (C-F) The gray lines were the co-linear blocks among the species, and the red lines highlighted the syntenic bHLH pairs between A. spinulosa and the species.

Supplementary Material 4

: Supplementary Fig.4. Predictive analysis of downstream target genes for AspbHLH80, AspbHLH120, and AspbHLH185. (A) AspbHLH80, (B) AspbHLH185, (C) AspbHLH120.

Supplementary Material 5

: Supplementary Table 1. The AspbHLHs primers of qRT-PCR used for detecting the expression patterns. Supplementary Table 2. Nucleotide sequences of primers used for gene cloning and vector construction Supplementary Table 3. Detailed information regarding bHLH gene family in five ferns. Supplementary Table 4. bHLH subfamily gene members in six species. Supplementary Table 5. The amino acid sequence of each conserved motif from each protein. Supplementary Table 6 − 1. Prediction of cis-regulatory elements in promoter regions of bHLH genes. Supplementary Table 6 − 2. Statistics on the quantitative relationship between cis-regulatory elements and bHLH genes. Supplementary Table 7. Ka, Ks and Ka/Ks of replication pairs of bHLH gene family in three ferns. Supplementary Table 8. Collinearity result among four ferns species. Supplementary Table 9. Expression of the AspbHLH family in different tissues (FPKM). (Pi: pith; Sb: sclerenchymatic belt; Xy: xylem; Ph: phloem; Le: leaf) Supplementary Table 10. Differential expression analysis of AspbHLH among five tissues (Pi: pith; Sb: sclerenchymatic belt; Xy: xylem; Ph: phloem; Le: leaf). Supplementary Table 11. GO enrichment analysis results of yellow module. Supplementary Table 12. KEGG enrichment analysis results of yellow module

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Huang, X., Fan, J., Liu, C. et al. Genome-wide identification of five fern bHLH families and functional analysis of bHLHs in lignin biosynthesis in Alsophila spinulosa. BMC Genomics 26, 357 (2025). https://doiorg.publicaciones.saludcastillayleon.es/10.1186/s12864-025-11522-z

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Keywords

BMC Genomics

ISSN: 1471-2164

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