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. 2025 May 6;25:594. doi: 10.1186/s12870-025-06452-7

Genome-wide analysis of the bHLH gene family in Spatholobus suberectus identifies SsbHLH112 as a regulator of flavonoid biosynthesis

Shuangshuang Qin 1,2, Ying Liang 1,2, Yueying Xie 1,2, Guili Wei 1,2, Quan Lin 1,2, Weiqi Qin 1,2, Fan Wei 1,2,✉
PMCID: PMC12054232  PMID: 40329176

Abstract

Spatholobus suberectus Dunn (S. suberectus), a medicinal herb from the Leguminosae family, is widely utilized in traditional medicine. The dried stem of S. suberectus demonstrates a variety of pharmacological effects, primarily attributed to its rich content of flavonoid compounds, such as catechin. The bHLH gene family serves diverse functions in plants, including regulating flavonoid biosynthesis, yet its specific function in S. suberectus remains uncertain. To address this, the sequenced genome of S. suberectus was leveraged for an extensive genome-wide analysis of the bHLH gene family. This analysis identified 156 SsbHLH genes, which were phylogenetically classified into 19 distinct subgroups. Of these, 153 genes were mapped across 9 chromosomes, while 3 remained unlocalized. Furthermore, genes within the identical subgroups displayed preserved exon-intron arrangements and motif patterns. Ka/Ks analysis further revealed that most duplicated genes have undergone purifying selection. A subset of 12 SsbHLH genes was found to be markedly associated with flavonoid content, including catechin, isoliquiritigenin, formononetin, and genistein. Among these, SsbHLH112, which strongly correlates with catechin levels, was shown to markedly elevate flavonoids and catechin accumulation when overexpressed in Nicotiana benthamiana. This overexpression also notably upregulated NbDFR and NbLAR, consistent with increased catechin production. These results elucidate the role of SsbHLH transcription factors in flavonoid biosynthesis, providing a basis for additional exploration of SsbHLH gene functions in S. suberectus.

Supplementary Information

The online version contains supplementary material available at 10.1186/s12870-025-06452-7.

Keywords: Spatholobus suberectus Dunn, Genome-wide analysis, Gene family, bHLH, Flavonoid biosynthesis

Introduction

The basic helix-loop-helix (bHLH) gene family ranks among the largest transcription factor (TF) families in eukaryotes [1], characterized by a conserved domain of nearly 60 amino acids, comprising two key motifs: a basic segment and a helix-loop-helix segment [2, 3]. Multiple investigations have shown the involvement of bHLH TFs in various plant developmental processes, including seed germination [4], flowering [5], and root hair development [6]. Furthermore, extensive research highlights their role in environmental responses, such as stress adaptation [7], plant mineral nutrition, and abiotic stress tolerance [8]. Substantial evidence also indicates the participation of bHLH TFs in phytohormone signaling and light response pathways [9, 10]. Additionally, bHLH genes serve a pivotal function in regulating the biosynthesis of secondary metabolites, encompassing terpenoids [11], alkaloids [12], phenolic acids [13], and flavonoids [14, 15].

With the increasing availability of complete and draft plant genomes, a significant number of bHLH transcription factors have been identified across various species, including important crops like maize [16], rice [17], and soybean [18], as well as model organisms such as Arabidopsis [19] and tobacco [20]. Medicinal plants, including Salvia miltiorrhiza [13], Panax ginseng [21], and Panax notoginseng [22], have also been found to contain these transcription factors, underscoring their widespread significance. Spatholobus suberectus Dunn (S. suberectus), a medicinal plant from the Leguminosae family, is extensively used in traditional medicine. Its dried stems exhibit various pharmacological properties, with flavonoids identified as the principal bioactive components [23, 24]. Among these, catechin has been demonstrated to enhance the growth of hematopoietic progenitor cells, while other flavonoids, encompassing genistein, isoliquiritigenin, and formononetin, have exhibited potential in cancer prevention and treatment [25–27]. While the biosynthetic pathways for these four flavonoids in S. suberectus have been well characterized [28], additional investigation is necessary to clarify the function of the bHLH gene family in regulating flavonoid biosynthesis within this species.

Research has demonstrated that the overexpression of bHLH transcription factors can precisely regulate the synthesis of specific secondary metabolites in plants [29, 30]. Thus, investigating the regulatory roles of SsbHLH genes in flavonoid biosynthesis offers promising avenues for boosting flavonoid production in S. suberectus. The availability of the published S. suberectus genome markedly facilitates the detection and examination of the bHLH gene family [31]. In this investigation, 156 SsbHLH genes were identified, followed by an in-depth analysis of their phylogenetic relationships, sequence characteristics, gene structure, motif composition, and chromosomal distribution. Correlation analysis revealed 12 candidate SsbHLH genes potentially involved in flavonoid biosynthesis, whose expression levels were quantified using RT-qPCR. Among these, SsbHLH112, associated with catechin biosynthesis, was selected for further functional analysis. This investigation presents a thorough overview of the SsbHLH gene family and offers crucial insights for future functional studies on their roles in flavonoid biosynthesis. Moreover, these discoveries will enhance our comprehension of the mechanisms governing flavonoid biosynthesis and its potential biotechnological applications.

Methodologies and materials

Plant materials

Fresh plant material from 16-year-old S. suberectus specimens was procured at the Guangxi Botanical Garden of Medicinal Plants (22°51’28"N, 108°22’2"E), Nanning, Guangxi, China. Samples were taken from stems, roots, leaves, flowers, and fruits. Additionally, Nicotiana benthamiana plants, used for Agrobacterium tumefaciens transformation, were grown under a controlled greenhouse environment with a 16/8 h light/dark cycle at 25 °C. Supplemental light of 4500 lx ensured optimal plant growth. All collected specimens were rapidly frozen using liquid nitrogen and maintained at -80 °C prior to RNA extraction and content analysis. To ensure result reliability and reproducibility, each sample contained three replicates.

Recognition of Spatholobus suberectus bHLH genes

The Hidden Markov Model (HMM) profile for the bHLH DNA-binding domain (accession number PF00010) was retrieved from the Pfam database (http://pfam.xfam.org/) [32]. This profile served as a query to conduct an HMM search within the S. suberectus genome utilizing HMMER 3.0’s default parameters [33] to identify the SsbHLH genes. To further confirm the accuracy of the identified bHLH protein sequences in S. suberectus, validation was performed using NCBI’s Conserved Domains Database (CDD) and the Simple Modular Architecture Research Tool (SMART), providing additional confidence in the sequence data.

Examination of SsbHLH gene sequences and structural features

All SsbHLH genes underwent examination utilizing the ProtParam tool (http://web.expasy.org/protparam/) to compute diverse characteristics such as isoelectric point, amino acid count, molecular weight, aliphatic index, and instability index. TBtools v1.045 software was employed to visualize gene structures, utilizing genomic sequences and coding regions to depict exon and intron lengths and numbers [34]. To detect conserved motifs within the SsbHLH protein sequences, the MEME v5.0.5 motif-based sequence analysis tool (http://meme-suite.org/) was applied [35].

Phylogenetic analysis of SsbHLH genes

Phylogenetic analysis involved comparing bHLH protein sequences from both S. suberectus and A. thaliana. Multiple sequence alignment was conducted utilizing MAFFT v7.427 with default parameters to ensure alignment efficiency and precision. A neighbor-joining (NJ) phylogenetic tree was generated employing Molecular Evolutionary Genetics Analysis (MEGA) 10.0 with the Poisson model, partial deletion, a 50% cutoff, and 1000 bootstrap iterations for robustness. The phylogenetic tree was visualized with FastTree [36]. Additionally, a separate phylogenetic tree was generated for all bHLH protein sequences using the same methodology. Integration of phylogenetic trees, conserved motifs, and gene structures into a comprehensive visual representation was achieved using TBtools v1.045 [34].

Genomic localization, collinearity analysis, and gene duplication of SsbHLH genes

The identified SsbHLH genes’ physical locations in S. suberectus were plotted on nine chromosomes utilizing MapGene2Chrom (MG2C), which generates physical gene maps in SVG format from input data [37]. Gene collinearity, both interspecific and intraspecific, was analyzed and visualized using the Multiple Collinearity Scan toolkit (MCScanX) and circos multiple synteny plot, respectively. The MCScanX parameters were set to a gap penalty of -1 and an E-value of 1e-10 [37]. To evaluate gene duplication events, nonsynonymous (Ka) and synonymous (Ks) substitution rates, along with the evolutionary constraint (Ka/Ks) between duplicated SsbHLH gene pairs, were computed utilizing KaKs_Calculator 2.0 [38]. Circos v0.69 was employed to visualize the synteny blocks of orthologous bHLH genes between S. suberectus and A. thaliana [39].

Expression analyses by RNA‑seq and correlation analysis of SsbHLH genes

The expression profiles of SsbHLH genes across diverse tissues, encompassing roots, stems, leaves, flowers, and fruits, were examined using transcriptomic data from prior research [28]. Gene expression intensities were ascertained in fragments per kilobase of exon model per million mapped reads (FPKM). Hierarchical clustering of the log2(FPKM+1) values was performed using Cluster 3.0 to group genes with similar expression patterns, and the results were visualized with Java TreeView [40]. The role of SsbHLH genes in flavonoid biosynthesis was further explored by measuring the levels of flavonoids such as catechin, isoliquiritigenin, genistein, and formononetin in various plant tissues based on previous research [28]. Correlations between SsbHLH gene expression and flavonoid content were examined using Spearman rank correlation analysis in R v3.6.2, with p-values below 0.05 considered statistically significant, indicating potential regulatory links between SsbHLH genes and flavonoid accumulation.

Agrobacterium transformation of Nicotiana benthamiana

To constitutively overexpress SsbHLH112 in N. benthamiana, primers listed in Supplemental Table S1 were utilized to amplify the complete CDS of SsbHLH112 from S. suberectus. The CDS was then inserted into the pBWA(V)HS vector using Eco31I and BsaI restriction enzymes, creating the Pro35S promoter-driven overexpression construct, Pro35S:SsbHLH112. Transformation of A. tumefaciens GV3101 cells with Pro35S:SsbHLH112 was performed utilizing the freezing method, and the transformed cells were employed to transform N. benthamiana plants. The genetic transformation protocols followed those previously outlined [41]. The transformation was confirmed via PCR using the Plant Direct PCR Kit (Nanjing Vazyme Biotech Co. Ltd., Nanjing, China) and gene-specific primers (Supplemental Table S1). Upon reaching a height of 8–10 cm and developing a robust root system, they were carefully uprooted, cleansed with water to remove any remaining agar, and then transplanted into soil. The SsbHLH112-transformed plants were subsequently cultivated in a greenhouse under controlled conditions until seeds were produced. Then, they were harvested and evaluated by cultivation on MS medium comprising 50 mg/L hygromycin B, with post-germination confirmation by PCR. The SsbHLH112 levels in the overexpressed plants were assessed through RT-qPCR, utilizing gene-specific primers from Supplemental Table S1.

Gene expression analyses by RT-qPCR

Gene expression analyses were conducted utilizing RT-qPCR following the previously outlined procedures [40], with gene-specific primers enumerated in Supplementary Tables S1 and S2. Three biological replicates were used, with each replicate consisting of distinct RNA samples derived either from three individual plants or a single plant. For normalization, 18 S and NbEF1α were chosen as the endogenous reference genes for S. suberectus and N. benthamiana, respectively [42, 43]. To confirm amplification specificity, a melting curve analysis was conducted. The quantification of gene expression levels was subsequently executed utilizing the 2−ΔΔCT methodology [44].

Flavonoid content and ultra-performance liquid chromatography-electrospray tandem mass spectrometry (UPLC-ESI-MS/MS) analysis

Flavonoid content was measured utilizing a kit from Suzhou Michy Biomedical Technology Co., Ltd. (Suzhou, China). The catechin standard (batch number SC8160) was acquired from Beijing Solarbio Science & Technology Co., Ltd. (Beijing, China). Ultra-performance liquid chromatography (UPLC) analysis was conducted using a Vanquish UPLC system (Thermo Scientific, USA) equipped with an ACQUITY UPLC HSS T3 column (100 mm × 2.1 mm, 1.8 μm) from Waters Corporation (Milford, MA, USA). The UPLC setup was interfaced with a TSQ Quantis™ Plus mass spectrometer (Thermo Fischer, USA), and integration of the primary isomer peak was accomplished utilizing TraceFinder 5.1 General Quan software (Thermo Fischer, USA). Catechin content was determined through UPLC-ESI-MS/MS, as detailed in our previous study [43].

Statistical analysis

All experimental data were obtained from at least three independent repetitions. Statistical relevance was ascertained utilizing Student’s t-test, with one-way ANOVA employed to assess significant differences across experimental conditions. The significance levels were defined as *p < 0.05 and **p < 0.01, denoting varying levels of confidence in the acquired outcomes.

Results

Recognition and analysis of bHLH gene family members in Spatholobus suberectus

The analysis of the S. suberectus genome revealed 156 candidate genes encoding SsbHLH domain-containing proteins. These SsbHLH proteins exhibited considerable variability in size, with amino acid sequences varying from 126 to 748. Their molecular masses spanned from 14.92 kDa (SsbHLH84) to 80.46 kDa (SsbHLH53), while their theoretical isoelectric points spanned from 4.64 (SsbHLH15) to 10.4 (SsbHLH27). Investigations into subcellular distribution further suggested that the majority of these proteins (144 out of 156, approximately 92.3%) were localized in the nucleus (Supplementary Table S3).

Phylogenetic analysis and categorization of bHLH genes in Spatholobus suberectus

The roles and evolutionary relationships of SsbHLH genes were investigated using an NJ phylogenetic tree generated with FastTree. This tree, which included 156 SsbHLH genes and 144 AtbHLH genes from Arabidopsis thaliana (Fig. 1), organized the SsbHLH genes into 19 subgroups (A1-A19), and the AtbHLH genes were divided into 21 subgroups (S1-S21) by reference [45]. Of these, 18 subgroups (comprising 148 SsbHLH genes) aligned with those in the AtbHLH phylogeny. Subgroups A5, A6, and A19 were the smallest, each containing only two members, while subgroup A7 was the largest, comprising 19 genes. Interestingly, subgroup A3 in S. suberectus failed to group with A. thaliana, suggesting an independent evolutionary trajectory for these genes in S. suberectus. Additionally, subgroups S12, S13, and S14 contained only AtbHLH genes, with no SsbHLH representation, indicating specific evolutionary divergences within the genome.

Fig. 1.

Fig. 1

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Phylogenetic comparison of SsbHLH proteins between Spatholobus suberectus (S. suberectus) and Arabidopsis thaliana (A. thaliana). The phylogenetic tree reveals distinct clades separating the SsbHLH subfamilies (depicted in various colors), resulting in 19 subgroups of the SsbHLH gene family (labeled A1–A19) and AtbHLH genes into 21 subpopulations (S1-S21)

Conserved gene structure and motif composition of SsbHLH genes

The exon-intron structures of the SsbHLH genes were examined to elucidate their structural diversity and potential functional implications (Fig. 2A, B). All 156 genes contained exons, with the exon counts ranging from 1 to 8, which collectively represented 98.7% of the total SsbHLH genes. The most common structure involved three exons (33 genes), while SsbHLH53 had 10 exons, and SsbHLH96 had 11 exons (Supplementary Table S3). To further explore structural diversity, 15 conserved motifs were predicted for all SsbHLH proteins (Fig. 2C, Supplementary Table S4). Motifs 1 and 2 were universally detected in all SsbHLH proteins, followed by motif 3 in 63 genes. Although motif composition varied across different subgroups, it was generally conserved within the same subgroup. For instance, motif 10 was exclusive to SsbHLH8, SsbHLH18, SsbHLH30, SsbHLH88, SsbHLH102, and SsbHLH137, all clustered within subgroup A14. Similarly, motif 8 was unique to SsbHLH3, SsbHLH55, SsbHLH113, SsbHLH115, and SsbHLH121, all found in subgroup A16 (Fig. 2C, Supplementary Table S3).

Fig. 2.

Fig. 2

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Gene structure and conserved motif analysis based on SsbHLH phylogenetic relationships. (A) Phylogenetic tree constructed using the NJ method with 156 SsbHLH protein sequences. (B) Gene structure analysis of SsbHLH genes., where orange and blue boxes represent exons and untranslated regions (UTRs), respectively, and black lines denote introns. (C) Conserved motifs in SsbHLH genes were identified using MEME Suite, with different colored boxes indicating distinct motifs. The scale bar of each SsbHLH gene is shown below each gene

Chromosomal location of SsbHLH genes

The chromosomal mapping of SsbHLH gene sequences revealed an uneven distribution across the genome. Of the 156 SsbHLH genes, 153 were distributed across 9 chromosomes: 20 genes each were located on chromosomes 1, 2, and 5; 12 on chromosome 3; 23 on chromosome 4; 37 collectively on chromosomes 6 and 7; and 21 on chromosomes 8 and 9. Chromosome 4 had the highest concentration of genes (23), succeeded by chromosomes 1, 2, and 5, each with 20 genes, while chromosome 9 had the fewest, with only 7 genes. Additionally, SsbHLH154 was located on scaffold_14, while SsbHLH155 and SsbHLH156 were mapped to scaffold_802 (Fig. 3).

Fig. 3.

Fig. 3

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Chromosomal mapping of SsbHLH genes. Out of 156 SsbHLH genes, 153 are mapped across 9 chromosomes, while 3 remain unassembled on scaffolds. Chromosome numbers are labeled at the top of each chromosome

Duplication events of SsbHLH genes

Gene duplication, encompassing both segmental and tandem duplication occurrences, serves a crucial function in the evolutionary process and diversification of gene families. By analyzing the collinearity of SsbHLH genes in S. suberectus, possible connections and duplication incidents were uncovered (Fig. 4). The study revealed 94 duplicated SsbHLH gene pairs, with the majority (82 pairs) resulting from segmental duplications, while 12 pairs were classified as tandem repeats. To further understand the evolutionary pressures acting on these duplicated genes, the ratios of non-synonymous to synonymous substitutions (Ka/Ks) were computed. The outcome indicated that all duplicated SsbHLH gene pairs had Ka/Ks ratios below one, suggesting that these genes have undergone purifying selection. This suggests that the duplicated genes are under strong evolutionary constraints, ensuring the preservation of their functions within the SsbHLH gene family in S. suberectus (Supplementary Table S5). To further explore the evolutionary history of the SsbHLH family, a comparative orthologous analysis was conducted between S. suberectus and the model species A. thaliana (Fig. 5). Previous research indicated that A. thaliana has five chromosomes with a genome size of 125 Mb [46]. This study identified 64 orthologs between S. suberectus and A. thaliana (Supplementary Table S6), providing valuable insights into the evolutionary conservation of the bHLH gene family across species.

Fig. 4.

Fig. 4

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Collinearity analysis of the bHLH gene family in S. suberectus. Synteny relationships between SsbHLH gene pairs are represented by curved lines of the same color

Fig. 5.

Fig. 5

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Collinearity analysis of between S. suberectus and A. thaliana bHLH genes. Syntenic gene pairs are connected by curved lines to show conserved relationships

Identification of the upstream regulatory SsbHLH genes of flavonoid biosynthesis

The expression patterns of SsbHLH genes were examined in diverse tissues, encompassing stem, leaf, and others, using previously collected transcriptome data (Fig. 6). Significant variations in SsbHLH gene expression were observed across these tissues (Supplementary Table S7). Several SsbHLH transcription factors, such as SsbHLH4, SsbHLH52, SsbHLH112, SsbHLH123, and SsbHLH133, exhibited specific expression in the stem. Notably, SsbHLH112 showed a 12.54-fold higher expression in the stem compared to the flower and a 4.42-fold higher expression compared to the leaf.

Fig. 6.

Fig. 6

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Expression profiles of SsbHLH genes in different S. suberectus tissues based on RNA-seq data

The flavonoid content also varied markedly across different plant parts, encompassing the root, stem, leaf, flower, and fruit. The stem exhibited the highest levels of flavonoids and catechin, while the fruit contained the most genistein. The root had the highest concentrations of isoliquiritigenin and formononetin [28]. To further investigate the regulatory mechanisms behind flavonoid biosynthesis, correlation analysis was performed using RNA-Seq data and flavonoid content measurements. Several SsbHLH genes were identified as being markedly associated with the levels of flavonoids, catechin, genistein, isoliquiritigenin, and formononetin (Fig. 7). Specifically, SsbHLH52, SsbHLH63, and SsbHLH133 were strongly correlated with flavonoids content, while SsbHLH112 and SsbHLH148 were closely linked to catechin concentration. Additionally, SsbHLH21, SsbHLH36, SsbHLH49, SsbHLH128, and SsbHLH136 were associated with genistein levels, and SsbHLH86 and SsbHLH139 were connected to formononetin and isoliquiritigenin contents.

Fig. 7.

Fig. 7

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Correlation between SsbHLH gene expression and flavonoid content. Spearman rank correlation analysis was performed in R (v3.6.2), with p < 0.05 (*) indicating statistical significance

Expression analyses of candidate SsbHLH genes by RT‑qPCR analysis

Further analysis of SsbHLH gene expression in flavonoid biosynthesis was conducted in five different tissues of S. suberectus (Fig. 8), which confirmed the transcriptome data. Genes such as SsbHLH52, SsbHLH63, SsbHLH112, and SsbHLH133, which were strongly correlated with flavonoids or catechin concentrations, showed particularly high expression in the stem. SsbHLH49, SsbHLH128, and SsbHLH136, linked to genistein, were predominantly expressed in the fruit, while SsbHLH86 and SsbHLH139, associated with formononetin and isoliquiritigenin, were highly expressed in the root. The differing flavonoid content across these tissues is probably influenced by the specific expression profiles of these related genes. The sustained high expression of certain SsbHLH genes implicated in flavonoid biosynthesis may account for the elevated flavonoid levels in S. suberectus tissues.

Fig. 8.

Fig. 8

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Expression analysis of 12 selected SsbHLH candidate genes across different S. suberectus tissues using RT-qPCR. Data are presented as mean ± SD from three biological replicates. Statistically significant differences (p < 0.05) between groups are indicated by different lowercase letters (a, b, c, d, e)

SsbHLH112-overexpression elevates flavonoids and Catechin contents and the expression of biosynthesis-related genes in transgenic Nicotiana benthamiana

In this study, SsbHLH112 exhibited a strong correlation with catechin concentration (Fig. 7) and was highly expressed in the medicinal stem tissue (Fig. 8), prompting further functional analysis. A p35S::SsbHLH112 vector was developed to investigate the role of SsbHLH112. Gene-specific primers were used for PCR (Fig. 9A) and RT-qPCR (Fig. 9B) to confirm the transgenic lines. Among the generated lines, three stable overexpression lines (OE2, OE4, OE12) in N. benthamiana were selected for detailed analysis. Phenotypic assessments revealed no notable distinctions between the transgenic strains and wild-type (WT) plants in terms of growth period, flowering time, or key morphological traits such as flower, leaf, and stem color (Fig. 9C). However, plant height increased markedly in lines OE4 and OE12 (Fig. 9D), while fresh weight and stem diameter remained comparable to the WT (Fig. 9E, F). Flavonoids and catechin contents were also measured in the WT and transgenic lines, with the contents of flavonoids increasing by 101.81%, 108.59%, and 224.24% in OE2, OE4, and OE12, respectively, compared to the WT (Fig. 9G). Similarly, catechin content rose by 219.98%, 80.31%, and 44.61% in these lines relative to the WT (Fig. 9H).

Fig. 9.

Fig. 9

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Overexpression of SsbHLH112 in Nicotiana benthamiana. (A) PCR validation of SsbHLH112 overexpression in transgenic lines. WT: wild type; OE1, OE2, OE4, OE9, OE10, OE11, and OE12: independent transgenic lines. (B) SsbHLH112 expression levels in wild type and SsbHLH112-overexpressing lines determined by RT-qPCR. Data are presented as mean ± SD (n = 3 plants per genotype). (C) Growth comparison between wild type and the three selected SsbHLH112 transgenic lines. (D) Plant height comparison between wild type and the three selected SsbHLH112 lines. (E) Stem diameter comparison between wild type and the three selected SsbHLH112 transgenic lines. (F) Fresh weight comparison between wild type and the three selected SsbHLH112 transgenic lines. (G) Flavonoids content in wild type and the three selected SsbHLH112 transgenic lines. (H) Catechin content in wild type and the three selected SsbHLH112 transgenic lines. *Indicates significant difference (P < 0.05); **indicates highly significant difference (P < 0.01)

RT-qPCR analysis (Fig. 10) of enzyme-encoding genes involved in the catechin synthesis pathway-NbPAL, NbC4H, Nb4CL, NbCHS, NbCHI, NbF3H, NbDFR, and NbLAR-revealed significant correlations between NbC4H, NbCHS, and NbF3H expression levels and flavonoids production in N. benthamiana (Supplementary Table S8). Additionally, the expression levels of NbDFR and NbLAR were strongly correlated with catechin content (Supplementary Table S8), aligning with the observed increases in catechin production in the transgenic lines. In particular, NbDFR expression in OE2, OE4, and OE12 was 22.2-, 6.2-, and 3.2-fold higher, respectively, than in WT, while NbLAR expression was elevated by 6.2-, 2.5-, and 1.6-fold in the same lines.

Fig. 10.

Fig. 10

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Comparison of transcriptional levels of genes involved in catechin biosynthesis between wild type (WT) and SsbHLH112 transgenic Nicotiana benthamiana using RT-qPCR. The enzymes analyzed include phenylalanine ammonia-lyase (PAL), cinnamate-4-hydroxylase (C4H), 4-coumarate CoA ligase (4CL), chalcone synthase (CHS), chalcone isomerase (CHI), flavanone-3-hydroxylase (F3H), flavanone-3’-hydroxylase (F3’H), dihydroflavonol 4-reductase (DFR), and leucoanthocyanidin reductase (LAR). Data are presented as mean ± SD (n = 3 plants per genotype). *Indicates a significant difference from WT (P < 0.05); **indicates a highly significant difference from WT (P < 0.01)

Discussion

Identification and phylogenetics of SsbHLH genes

The bHLH gene family serves a vital function in various physiological processes and has been discovered in numerous plant species. In this investigation, 156 SsbHLH genes were detected and characterized in the S. suberectus genome. Although this number is lower than that found in Glycine max (319) and Medicago sativa (469), both of which also belong to the Leguminosae family [47, 48], it is significant given the genome size of S. suberectus (798 Mb), which is smaller than G. max (1100 Mb) but slightly larger than M. sativa (793.2 Mb) [31, 49, 50]. This indicates that gene family size is not solely dependent on genome size but is also influenced by the species’ evolutionary history. The 156 SsbHLH proteins were classified into 19 subgroups, a number lower than in Hordeum vulgare [51], Panax ginseng [52], and Cinnamomum camphora [53], yet higher than in Zea mays [54] and Aquilaria sinensis [55]. Notably, subgroups A3 only included SsbHLH genes but no AtbHLH genes, which may be attributed to the loss of genes during evolution in A. thaliana. Furthermore, subgroups S12, S13, and S14 contained only AtbHLH genes, with no SsbHLH genes present, suggesting that the bHLH genes were either acquired by A. thaliana or lost by S. suberectus during evolution. Such species-specific gains and losses of bHLH genes may contribute to functional divergence.

Gene structure and motif analysis of bHLH genes in Spatholobus suberectus

Further examination of conserved motifs and gene structures supported the phylogenetic relationships within the SsbHLH transcription factor family. For instance, motif 8 was unique to subgroup A16, and motif 10 was exclusive to subgroup A14. Most SsbHLH genes within the same subgroup exhibited similar gene structures and conserved motifs, indicating they likely share similar biological functions [56]. For example, AtbHLH genes in subgroup XIII, including AtbHLH4 (At4g17880), AtbHLH5 (At5g46760), and AtbHLH6 (At1g32640), have been shown to regulate flowering time through the JA pathway [57, 58]. Additionally, motifs 1 and 2 were consistently found together in all SsbHLH proteins, while motif 3 was present in most SsbHLH genes, suggesting a high degree of evolutionary conservation. This points to their essential role in maintaining the DNA-binding capacity of SsbHLH proteins [59]. The SsbHLH genes exhibited a wide range of exon numbers, from 1 to 11, with 13 genes containing only one exon and SsbHLH96 being the only gene with more than ten exons. This variability may reflect ongoing evolutionary processes within the SsbHLH gene family.

Gene duplication of bHLH genes in Spatholobus suberectus

Gene duplication, including whole-genome duplication (WGD), segmental duplication, and tandem duplication, is a key driver in the expansion of gene families and the creation of new genes in plants [60]. Previous studies have identified three WGD events in G. max and two in S. suberectus [28], which likely contribute to the markedly higher number of GmbHLH genes (319) compared to SsbHLH genes (156). Gene duplication serves as a vital mechanism in fostering species diversity and is essential for facilitating plant adaptation to shifting environmental conditions [61, 62]. Segment duplication and tandem duplication are the two main gene duplication modes leading to gene family expansion [63]. In S. suberectus, 82 segmental duplications and 12 tandem duplications of bHLH genes were detected, suggesting that segmental duplication was the primary driver of SsbHLH gene expansion. Repetitive bHLH genes also shared a similar structure and motif composition [64]. Ka/Ks analysis revealed that most SsbHLH genes have undergone purifying selection, suggesting high evolutionary conservation. Comparative orthologous analysis between A. thaliana and S. suberectus demonstrated significant collinearity, indicating conserved gene arrangements and synteny, likely due to shared evolutionary history.

Candidate SsbHLH genes markedly associated with flavonoid synthesis

Previous research has highlighted the role of bHLH genes in regulating flavonoid biosynthesis in various plants. For instance, MYCA1 regulates flavonoid accumulation in Vitis vinifera [65], PsbHLH1 in Paeonia suffruticosa activates the expression of PsDFR and PsANS to positively regulate flavonoid biosynthesis [66], and EbbHLH80 in Erigeron breviscapus regulates the expression of several structural genes implicated in the flavonoid biosynthesis pathway, including PAL, C4H, CHS, 4CL, CHI, DFR, and ANS, to enhance flavonoid accumulation [67]. In this study, 12 candidate SsbHLH genes were found to be markedly correlated with flavonoid synthesis, although their specific functions require further investigation. Overexpression of SsbHLH112 in N. benthamiana led to marked increases in flavonoids and catechin levels. Compared to wild-type controls, SsbHLH112 overexpression markedly upregulated NbDFR and NbLAR, aligning with the observed rise in catechin content. These findings suggest that SsbHLH112 may promote catechin accumulation by regulating NbDFR and NbLAR expression. Overall, this study demonstrates the role of SsbHLH112 as a regulator of catechin biosynthesis in N. benthamiana, but further gene function validation experiments in S. suberectus are needed.

Conclusion

This research conducted an extensive genome-wide analysis of the SsbHLH gene family in S. suberectus, identifying 156 SsbHLH genes, which were classified into 19 subgroups. Of these, 153 genes were localized on 9 chromosomes. Genes within the identical subgroup exhibited conserved motif structures and exon-intron similarities, supporting the phylogenetic findings. Synteny analysis demonstrated that the growth of the SsbHLH gene family was predominantly driven by segmental duplications. Ka/Ks analysis further suggested that these genes experienced purifying selection. Notably, 12 SsbHLH genes were strongly associated with the biosynthesis of flavonoids, catechin, isoliquiritigenin, formononetin, and genistein. Overexpression of SsbHLH112, which showed a significant correlation with catechin concentration, substantially enhanced flavonoids and catechin levels in N. benthamiana. Compared to wild-type controls, SsbHLH112 overexpression markedly upregulated the expression of NbDFR and NbLAR, corresponding with the increase in catechin content. These results shed light on the regulatory role of SsbHLH transcription factors in flavonoid biosynthesis and establish a basis for additional exploration of SsbHLH gene functions in S. suberectus.

Electronic supplementary material

Below is the link to the electronic supplementary material.

Supplementary Material 1 (378.4KB, xlsx)
Supplementary Material 2 (10.4MB, tif)

Acknowledgements

Thanks to all who participated in this study.

Author contributions

S.Q. and W.F. planned and designed the research. S.Q., Y.L., Y.X. and Q.L. collected and analyzed the data. G.W., Q.L. and W.Q. performed experiments. S.Q. wrote the manuscript. S.Q. and W.F. revised the manuscript.

Funding

This study was fnancially supported by the National Natural Science Foundation of China [grant number: 82160723] and the Natural Science Foundation of Guangxi Zhuang Autonomous Region [grant number: 2025GXNSFAA069354 and 2023GXNSFAA026487].

Data availability

Data is provided within the manuscript or supplementary information files.

Declarations

Ethics approval and consent to participate

Not applicable.

Consent for publication

Not applicable.

Competing interests

The authors declare no competing interests.

Footnotes

Publisher’s note

Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.

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Supplementary Materials

Supplementary Material 1 (378.4KB, xlsx)
Supplementary Material 2 (10.4MB, tif)

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