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Article

Genome-Wide Identification of the bHLH Gene Family and Expression Analysis in Anthocyanin Synthesis in Lagerstroemia indica Leaves

by
Lu Feng
1,†,
Yanhong Guo
2,†,
Xu Han
3,
Aiqin Ding
1 and
Jing Shu
1,*
1
College of Forestry Engineering, Shandong Agriculture and Engineering University, Jinan 250100, China
2
College of Horticulture and Landscape Architecture, Zhongkai University of Agriculture and Engineering, Guangzhou 510225, China
3
College of Agricultural Science and Technology, Shandong Agriculture and Engineering University, Jinan 250100, China
*
Author to whom correspondence should be addressed.
†
These authors contributed equally to this work.
Horticulturae 2025, 11(10), 1219; https://doi.org/10.3390/horticulturae11101219
Submission received: 20 August 2025 / Revised: 29 September 2025 / Accepted: 5 October 2025 / Published: 10 October 2025
(This article belongs to the Section Genetics, Genomics, Breeding, and Biotechnology (G2B2))
Browse Figures
Versions Notes

Abstract

The basic Helix-Loop-Helix (bHLH) transcription factor family is crucial for plant growth, development, and stress response regulation. Despite previous studies on the bHLH gene family in Lagerstroemia indica, many bHLH genes remain unidentified, hindering further research on LibHLHs. Here, we identified 150 LibHLHs from the genome of L. indica and categorized them into 12 subfamilies (comprising 25 subgroups) showing conservation within subgroups. Cis-acting element analysis suggests roles in plant development, and responses to light, hormones, and stress. Examination of gene expression patterns highlighted the potential involvement of specific genes, such as LibHLH25 in subgroup IIIf, LibHLH68, LibHLH106, and LibHLH142 in subgroup IIIb, and LibHLH112 in subgroup VIIa, in anthocyanin biosynthesis in leaves of L. indica. This investigation enhances our comprehension of the complexity of the bHLH gene family and highlights the potential roles of LibHLHs in anthocyanin biosynthesis in L. indica, offering valuable insights for future genetic breeding endeavors.

1. Introduction

Transcription factors are pivotal in regulation of eukaryotic growth and development by overseeing the expression of target genes [1]. Among these, bHLHs are crucial in both plant and animal systems. The conserved bHLH domain, approximately 60 amino acids in length, comprises two key segments: the N-terminal DNA binding region and the C-terminal helix-loop-helix (HLH) region [2,3]. This conserved domain enables bHLH to bind to the E-box or G-box on gene promoters and form dimers, thus modulating gene expression across diverse signaling pathways [1,4].
In Arabidopsis thaliana, 162 bHLHs have been identified and categorized into 12 subfamilies [5]. Advances in sequencing technology have enhanced genomic databases for numerous important plant species, leading to whole-genome investigations on bHLH transcription factors across various plants. The bHLH genes have been identified in several plant species such as rice [6], Secale cereale [7], Ginkgo biloba [8], Prunus sibirica [9], carrot [10], and peach [11]. Through evolutionary relationships, conserved domains, and reference to the AtbHLH family classification, the bHLH transcription factor family is commonly subdivided into 15 to 25 subfamilies. bHLH genes are crucial in processes such as plant development, light signal transduction, abiotic stress response [12,13,14,15,16,17], and crosstalk among hormone signals [18,19,20,21,22]. Moreover, bHLH serves as a key transcription factor regulating flavonoid biosynthesis [23,24,25,26]. In A. thaliana, bHLH directly involved in anthocyanin synthesis are clustered within the IIIf subfamily, forming ternary complexes with R2R3-MYB and WD40 proteins to control anthocyanin and proanthocyanidin production [27,28] and directly modulate the expression of anthocyanin biosynthesis genes [29]. In other species, the subfamily IIIf genes also play an important role in anthocyanin synthesis [15,30,31].
L. indica is highly valued for its ornamental characteristics, including vibrant flower colors, prolonged blooming period, and graceful tree structure, making it a popular tree species in Chinese horticulture. Recently, cultivars with colorful leaves have been developed, significantly enhancing their aesthetic appeal. Research on anthocyanin biosynthesis in leaves of L. indica has revealed distinct anthocyanin profiles in purple leaves compared to petals. The transcription factors LiHY5 and LiMYB75 may be implicated in leaf anthocyanin synthesis [32,33]. Although the bHLH transcription factor family has been identified in a variety of plant species, limited research has been conducted on L. indica. This investigation aimed to identify the bHLH family in L. indica using available whole-genome and transcriptome data from crape myrtle variants with different leaf colors. The results provide a foundation for further investigation into bHLH’s role in the biosynthesis of anthocyanins in L. indica.

2. Materials and Methods

2.1. Identification of bHLH Gene Family in L. indica

The genomic data for L. indica was retrieved from the National Genomics Data Centre (NGDC) (https://ngdc.cncb.ac.cn/gsa/, accessed on 11 February 2025, accession: PRJCA013427), with A. thaliana serving as the reference species. Information on the bHLH gene family of A. thaliana was sourced from The Arabidopsis Information Resource database (https://www.arabidopsis.org/, accessed on 11 February 2025). Seventy AtbHLH protein sequences were utilized as templates for BLAST searches in TBtools (v2.357) to eliminate redundant sequences. The Hidden Markov Model (HMM, PF00010) for the bHLH domain was sourced from the InterPro database (https://www.ebi.ac.uk/interpro/, accessed on 13 February 2025). Subsequently, HMMER 3.0 software, with default parameters, identified the LibHLH protein sequence in L. indica. Furthermore, NCBI CDsearch (https://www.ncbi.nlm.nih.gov/Structure/cdd/wrpsb.cgi/, accessed on 15 February 2025) was used to validate the completeness of the bHLH domain and exclude incomplete genes. Expasy (https://web.expasy.org/protparam/, accessed on 24 February 2025) predicted the physicochemical properties of LibHLHs, including protein amino acid quantity (aa), theoretical isoelectric point (pI), molecular weight (MV), instability coefficient, and hydrophilic index. WoLF PSORT (https://wolfpsort.hgc.jp/, accessed on 24 February 2025) was employed to predict the subcellular localization of the LibHLH family.

2.2. Phylogenetic Analysis of LibHLH Proteins in L. indica

Cluster analysis was performed on 150 LibHLH proteins and 70 AtbHLH proteins sequences using the Neighbor Joining method in MEGA X64. The analysis was validated with 1000 bootstrap replicates. The final tree visualization was generated using iTOLv6 (https://itol.embl.de/, accessed on 1 March 2025).

2.3. Analysis of Conserved Motifs and Cis-Acting Elements of Promoters

We utilized the MEME (https://meme-suite.org/meme/tools/meme/, accessed on 5 March 2025) to identify conserved motifs within the LibHLH protein, specifying a maximum of 10 motifs while maintaining default settings for other parameters. The conservative domain structure of LibHLHs was predicted using the NCBI CD-Search tool (https://www.ncbi.nlm.nih.gov/Structure/bwrpsb/bwrpsb.cgi/, accessed on 4 April 2025). Visualization was achieved with the “Gene Structure View” module in TBtools. Furthermore, we predicted cis-acting elements within the 2000 bp promoter region upstream of the LibHLH gene using PlantCARE online software (http://bioinformatics.psb.ugent.be/webtools/plantcare/html/, accessed on 1 April 2025).

2.4. Chromosomal Mapping and Collinearity Analysis of LibHLHs in L. indica

The genome data of L. indica was utilized to extract the chromosome localization information of the LibHLH gene family. Subsequently, TBtools software was employed to construct a chromosomal localization map of the LibHLH gene family in L. indica, followed by an analysis of its distribution. Furthermore, the collinearity of bHLH genes between L. indica, A. thaliana, and Punica granatum was analyzed and visualized using the “MCScanX” tool of TBtools.

2.5. Protein–Protein Interaction Network Prediction

To predict the interaction between LibHLHs proteins, we used AtbHLHs as a reference protein to construct an interaction network using an online STRING (https://cn.string-db.org/, accessed on 28 April 2025).

2.6. Gene Expression Analysis of LibHLHs

The RNA sequencing data utilized in this study were obtained from our prior investigation (NCBI login Number: PRJNA610678). Materials for sequencing comprised three-year-old potted cuttings of L. indica ‘Ebony Embers’ and ‘Arapahoe.’ These were cultivated outdoors at the China National Engineering Research Center for Floriculture (CNERCF) in Beijing, China (40°17″ N, 116°39″ E), and were uniformly pruned in August 2020. Leaves at 5-day (S1), 15-day (S2), and 30-day (S3) stages of germination were collected from sunny branches on clear mornings between 9 and 10 a.m. for the experiments. Transcriptome sequencing was conducted using the Illumina HiSeq 4000 platform by Gene Denovo Biotechnology Co. (Guangzhou, China). Transcript assembly was annotated for gene function utilizing public databases, including GO (Gene Ontology), KEGG (Kyoto Encyclopedia of Genes and Genomes), applying an e-value threshold of ≤10−5. Gene expression levels were quantified using the RPKM method. Differentially expressed genes across various comparison groups were identified based on the criteria of DFR threshold < 0.05, |log2FC| > 1 and p-value < 0.05.
Total RNA was extracted from ‘Ebony Embers’ and ‘Arapahoe’ leaves at stages S1, S2, and S3 using the EASYspin Plus Composite Plant RNA Kit (Aidlab, Beijing, China). First-strand cDNA synthesis was performed with the TransScript® One-Step gDNA Removal and cDNA Synthesis SuperMix (TransGen, Beijing, China). Real-time fluorescence quantification was conducted with TB Green Premix Ex Taq II (Tli RNase H Plus) (Takara, Beijing, China), followed by Quantitative real-time PCR (qRT-PCR) analysis as previously described [34]. The gene relative expression levels were quantified using a PCR instrument (CFX connect, Bio-Rad, Hercules, CA, USA). The thermal cycling protocol consisted of an initial denaturation step at 95 °C for 30 s, followed by 40 amplification cycles, each comprising 5 s at 95 °C and 30 s at 60 °C. The melting curve was recorded by increasing the temperature from 60 °C to 95 °C in 0.5 °C increments every 5 s. The internal reference gene EF-1α (GenBank ID: MG704141) was utilized, and gene expression levels were calculated using the 2−ΔΔ method [35]. Each reaction was replicated three times. The primers used were designed by Primer Premier 5 with the annealing temperature at 60 ± 2 °C and subjected to blast specificity testing in L. indica to ensure target specificity. The qRT-PCR amplification efficiency was required to be between 90% and 110%, and a single peak in the melting curve analysis was necessary to confirm amplification specificity. The primer sequences are listed in Table S1.

3. Results

3.1. Identification and Protein Characteristics Analysis of the bHLH Gene Family in L. indica

Blast and HMMsearch were employed to screen potential bHLH gene family members in L. indica. After removing genes with incomplete conserved domains, 150 bHLH genes were finally identified and labeled as LibHLH1 through LibHLH150 based on chromosomal location. Analysis of the bHLH proteins revealed amino acid lengths ranging from 143 aa (LibHLH54) to 835 aa (LibHLH114), with molecular weights spanning 15.67 kDa (LibHLH54) and 90.75 kDa (LibHLH114). The theoretical isoelectric points (pI) varied from 4.77 (LibHLH1) to 9.66 (LibHLH117). Instability indices ranged from 39.8 (LibHLH90) to 77.48 (LibHLH71), with 99.4% (149 proteins) exceeding an index of 40, indicating instability. Hydrophilicity index predictions showed one protein with a value above 0, while the rest were below 0, suggesting most LibHLH proteins are hydrophilic. In the prediction results of subcellular localization, 135 LibHLHs of L. indica were nuclear proteins, accounting for 90.00%, which was the largest proportion. Followed by chloroplasts (8, 5.3%), cytoplasm (2, 1.3%), vacuoles (2, 1.3%), plasma membranes (2, 1.3%), and Golgi apparatus (1, 0.7%) (Table S2).

3.2. Phylogenetic Analysis of the bHLH Gene Family in L. indica

We constructed a phylogenetic tree to examine the grouping and evolutionary trends of LibHLHs, incorporating bHLHs from L. indica and A. thaliana. Following the subgroup categorization of A. thaliana [1], we identified 150 LibHLHs distributed across 12 subfamilies, encompassing 25 subgroups, which closely align with the bHLH protein classification in A. thaliana (Figure 1). Among them, subfamily XII was the most populous, comprising 21 LibHLHs proteins, representing 14% of the total. Conversely, subgroups VIIb and VIIIa each encompassed only one LibHLH protein, constituting the smallest proportion at 0.7%. Other subgroups with a relatively large number of proteins were Ia and X, with 15 and 14 LibHLHs, respectively. In Arabidopsis thaliana, the IIIf subgroup mainly regulates the flavonoid pathway [36], and LibHLH25, LibHLH50, LibHLH57, LibHLH61, and LibHLH62 were clustered in this subgroup.

3.3. Gene Structure and Cis-Acting Elements of the bHLH Gene Family in L. indica

The evolutionary relationships and structural similarities within the LibHLH gene family were investigated through a comprehensive analysis incorporating phylogenetic data, gene structure, and motif analysis. Our findings, as illustrated in Figure 2B, revealed that each LibHLH protein contained between 2 and 7 motifs, with motifs 1 and 2 being universally present and closely linked to the conserved bHLH domain. Distinct motifs were identified across different subgroups: both subgroups X and XI featured motif 8, subgroup Ia included motif 10, and subgroups IIId, IIIe, and IIIf shared motifs 5, 6, and 9. The consistent motif composition within each subgroup suggests that LibHLHs in the same subgroup may perform similar functions.
Conserved domain analysis (Figure 2C) identified nine types of conserved domains within the LibHLH gene family, with the bHLH domain present in every LibHLH protein. Certain subgroups exhibited specific conserved domains; for example, subgroups IIId, IIIe, and IIIf consistently featured the bHLH-MYC_N domain at the N-terminus, while nearly all members of subgroup Vb possessed an ACT domain at the C-terminus. The uniformity in domain types and numbers within each group indicates convergent evolutionary relationships.
Analysis of gene structure (Figure 2D) revealed that the LibHLH gene family contained 1 to 18 coding sequences (CDSs), with 13 genes lacking introns. Within the same subfamily, CDS and intron number, length, and location were consistent. For instance, subgroup IVc had 4 introns, subgroup VIIIb had none, subgroup VIIIc generally had 4, and subgroup Vb typically had 1 intron.
To elucidate the functions and transcriptional features of LibHLHs, we conducted an analysis of cis-acting elements present in the 2000 bp promoter region upstream of their state codon. Our findings revealed that, beyond the typical promoter elements (TATA-box, CAAT-box, A-box, 3-AF3 binding site), the elements predominantly included those responsive to light, growth and development, biotic and abiotic stresses, and phytohormones. All LibHLHs contained light-responsive elements, while 120 LibHLHs harbored elements associated with growth and development, linked to various tissues and developmental processes such as meristem, seed, endosperm, root, leaf, cell cycle, and circadian rhythm. Phytohormone-responsive elements encompassed five categories: abscisic acid, gibberellin, methyl jasmonate, and salicylic acid, with abscisic acid-responsive elements being the most prevalent (143), and salicylic acid-responsive elements being the least common (64). Additionally, five types of stress-responsive elements were observed: anoxia, drought, low temperature, stress defense, and wounding. Anoxia-related elements were the most abundant (116), whereas wounding-responsive elements were the least (10). Furthermore, elements linked to flavonoid biosynthesis were identified in 15 members, not within the subgroup IIIf but distributed across subgroup XII (6), Ia (3), IIIb (3), IIId (2), IIIe (2), IIIa (1), and X (1) (Figure S1, Table S3).

3.4. Chromosomal Distribution and Collinearity Analysis of LibHLH Genes

Chromosomal localization revealed that 150 LibHLH genes are unevenly distributed across all 24 chromosomes. Chromosome 6 harbored the highest number, with 14 genes (9.33%), followed by chromosome 4 with 12 genes (8%). Chromosomes 5, 8, and 11 each contained 10 genes. In contrast, chromosome 19 had the fewest, with just 1 gene (0.67%) (Figure 3).
To elucidate the evolutionary dynamics of the LibHLH gene family, we conducted collinearity analyses on the L. indica genome. In the LibHLH gene family, we identified 88 segmental duplication pairs, encompassing 109 genes across 22 chromosomes, excluding chromosomes 9 and 21. These genes belonged to 21 subgroups, excluding II, IIIa, VIIb, and VIIIa. The proportion of segmentally duplicated LibHLH genes per subgroup ranged from 33% to 100%, with 17 subgroups exceeding 67%, and 6 subgroups entirely comprising segmentally duplicated genes. Notably, except for the LibHLH69–LibHLH132 pair, all gene pairs were confined to the same subgroup (Figure 4, Table S4). Furthermore, we identified four tandem duplications on chromosomes 5, 6, 14, and 15, involving eight genes. Tandem pairs LibHLH31 and LibHLH32, LibHLH48 and LibHLH49, LibHLH97 and LibHLH98, and LibHLH112 and LibHLH113 were clustered in subfamilies Ib, II, IVa, and VIIa, respectively. These findings suggest that the LibHLH gene family predominantly expands through segmental duplication.
We constructed collinearity maps for L. indica with A. thaliana and P. granatum (Figure 5), identifying 169 and 196 collinear gene pairs, respectively. This suggests a closer evolutionary relationship between L. indica and P. granatum. Notably, 79.9% of the collinear pairs between L. indica and A. thaliana involved a single A. thaliana gene corresponding to multiple LibHLH genes, with up to five LibHLH genes linked to one AtbHLH gene. A similar pattern was observed between L. indica and P. granatum, where 84.22% of the collinear gene pairs showed one PgbHLH gene corresponding to multiple LibHLH genes, with up to 6 LibHLH genes matching a single PgbHLH gene. These findings indicate extensive gene duplication events during the evolution of L. indica.

3.5. Protein–Protein Interaction Analysis of LibHLH Genes

We explored the interaction network of LibHLH proteins (Figure 6) based on homologous AtbHLHs. LibHLH58, LibHLH124, LibHLH128, and LibHLH149 showed no interactions with other LibHLH proteins, while LibHLH82 and LibHLH134 interacted exclusively with each other. The remaining LibHLHs exhibited complex interaction patterns. Notably, AtbHLH112 (LibHLH47), AtbHLH106 (LibHLH66, LibHLH85), AtbHLH30 (LibHLH30, LibHLH136, LibHLH138, LibHLH146), and AtbHLH35 (LibHLH1, LibHLH150) interacted with multiple bHLH proteins, playing roles in various abiotic stress responses [37]. These findings suggest that several LibHLH proteins contribute to plant development and stress resistance by forming protein complexes.

3.6. Expression Patterns of LibHLHs in L. indica with Different Leaf Colors

We employed existing transcriptome data to examine the expression profiles of LibHLH genes in leaves. Transcriptome data from leaves at different developmental stages were examined in the cultivar ‘Arapahoe’ with green leaves and the cultivar ‘Ebony Embers’ with purple leaves. The results showed that 28 LibHLHs had extremely low expression levels in leaves, mainly belonging to Ib (7, 78%), VIIIc (7, 70%), IVa (3, 50%), and VIIIb (3, 50%). Excluding these 28 genes, the remaining 122 genes could be classified into groups A, B, C, D, and E according to their expression patterns (Figure 7A). Notably, members of the same subgroup exhibited different expression patterns. Group A genes exhibited consistently low expression throughout green leaf development, whereas their expression increased progressively in purple leaves. The expression of Group B genes increased significantly in the late stage of green leaf development; although expression increased in purple leaves, it remained lower than in green leaves. The gene expression level of Group C decreased with development, yet was higher in purple leaves, particularly at the S1 stage. Group D genes demonstrated a consistent decline in expression level across development, with similar levels in both leaf types. Group E genes also showed a gradual decrease, but expression level was higher in green leaves, notably at the S1 stage.
We observed that the expression patterns of the key anthocyanin structural genes LiCHS, LiDFR, and LiANS were similar to those of Group C genes, as evidenced by previous research and the current findings (Figure 7B,C). Notably, LibHLH4, LibHLH25, LibHLH48, LibHLH49, LibHLH54, LibHLH68, LibHLH104, LibHLH106, LibHLH112, LibHLH137, LibHLH142, and LibHLH144 showed a strong correlation with the expression levels of these structural genes. These 12 LibHLHs spanned nine subgroups: LibHLH25 belonged to subgroup IIIf linked to flavonoid synthesis, while LibHLH68, LibHLH106, and LibHLH142 belonged to the subgroup IIIb, and LibHLH112 to the subgroup VIIa. The expression levels of the examined genes were generally elevated in purple leaves, particularly at the S1 developmental stage. Most genes exhibited a downward trend in expression during leaf development, with some exceptions. LibHLH48 displayed an upward-then-downward pattern in purple leaves, while LibHLH112 showed a downward-then-upward trend in purple leaves. The results of qRT-PCR were highly consistent with the transcriptome sequencing data. (Figure 8).

4. Discussion

The bHLH transcription factor family, among the largest in plants, plays a crucial role in regulating growth, development, and stress resistance [15,38]. To date, genes belonging to the bHLH TF family have been extensively investigated in various plant species such as A. thaliana (162) [39], Oryza sativa (183) [40], Zea mays (231) [40], Triticum aestivum (571) [40], Ananas comosus (121) [41], Secale cereale (220) [7], Ginkgo biloba (85) [8], and Hordeum vulgare (141) [42], etc. The bHLH transcription factor family exhibits substantial numerical variation across species due to evolutionary processes [15]. Earlier research identified 79 bHLH genes in L. indica using petal transcriptome data from different developmental stages, yet some genes remain unidentified [43]. Further advancements are required in the identification and research of LibHLHs. This study enhances the bHLH family gene library of L. indica and analyzes their transcriptional levels during anthocyanin synthesis in the leaf.
This study identified 150 bHLH genes from the L. indica genome, showing significant variability in protein length and theoretical isoelectric points, likely due to gene duplication events. This complexity is also observed in other species. The number of LibHLH genes is intermediate relative to those reported for various plant genomes. However, no clear relationship has been found between bHLH family size and genome size or chromosome number across species [15,44]. LibHLH genes are distributed across all L. indica chromosomes, but the gene count on each chromosome does not correlate with chromosome size. Notably, chromosomes 4, 5, 6, 8, and 11 have more genes despite not being the largest. This uneven distribution, seen in species like maize [45], tomato [44], rice [6], Spatholobus suberectus [46], and Gastrodia elata [47], may result from irregular chromosome segment duplication events.
A phylogenetic analysis of the bHLH gene family in L. indica was conducted based on the classification of AtbHLH genes. This, along with findings from other species, suggests that bHLH proteins in these subfamilies likely share a common ancestor. The diversity within the bHLH gene family appears to have existed since the advent of land plants, and the subsequent diversification of bHLH proteins across species has contributed to the complexity in their classification [15]. Notably, the LibHLH gene family appears to have retained the protein functions represented by the various AtbHLH subfamilies, without apparent loss during evolution.
Analysis of the gene structures, conserved domains, and motifs of 150 LibHLH genes reveals that LibHLH proteins within the same subfamily exhibit strong conservation across these aspects. Among the 10 identified motifs, motif1 and motif2 consistently appear adjacent to each other across all genes, suggesting they form part of the bHLH conserved domain. Each subfamily also shows distinct characteristics; for instance, subgroups IIId, IIIe, and IIIf all feature motifs 5, 6, and 9 near the N-terminus, a pattern also observed in A. thaliana. This conserved region is unique to plant bHLH proteins, underscoring the specificity of the protein structures in these subgroups. Studies of gene structure reveal that genes within the same subfamily exhibit similarities in exon number and distribution, indicating strong evolutionary and functional conservation. Interestingly, some individual LibHLH genes possess an exceptionally large number of exons, potentially indicative of functional divergence [48]. Cis-acting elements in the LibHLHs promoter can be grouped into four main categories: light—responsive elements, phytohormone—responsive elements, biotic and abiotic stress—responsive elements, and growth and development—related elements. Predominantly, light-responsive, abscisic acid-responsive, and MeJA-responsive elements are most abundant, aligning with observations in other plant species like Magnolia sieboldii [46], plum [49], and Rosa persica [50]. Notably, the LibHLH promoter regions commonly harbor multiple cis-acting elements, underscoring their involvement in diverse physiological and developmental processes. Fifteen LibHLHs contain elements related to flavonoid synthesis regulation, yet they do not fall within the subgroup IIIf, typically associated with flavonoid synthesis. Instead, they are primarily categorized under subgroups XII, Ia, and IIIb. In Arabidopsis, these subgroups are linked to brassinosteroid-mediated growth and development [14,51,52], stomatal development [53], and low-temperature response [54]. Notably, ICE1 in subgroup IIIb is implicated in flavonoid accumulation during cold acclimation [55]. Although these genes do not show consistency with the expression patterns of anthocyanin structural genes, whether they are involved in the anthocyanin synthesis in L. indica requires further investigation.
Gene duplication is a pivotal factor in genome and genetic system evolution, with large-segment and tandem duplications primarily driving plant gene family expansions [56]. Our analysis of gene duplication events in the LibHLH gene family revealed that the expansion of LibHLHs in L. indica was primarily driven by segmental duplication. The LibHLHs obtained through these duplication events are highly conserved and belong to the same subgroup, indicating their pivotal role in the evolution of the bHLH gene family in L. indica, potentially enhancing environmental adaptability and regulating growth and development. Furthermore, collinearity analysis indicated a close genetic relationship between L. indica and P. granatum, with a single PgbHLH corresponding to multiple LibHLHs. These homologous genes may have contributed to the evolution of the LibHLH gene family.
This study conducted a correlation analysis between LibHLHs and the key structural genes LiCHS, LiANS, and LiDFR involved in anthocyanin synthesis in L. indica. Results indicated that there was a strong correlation between the expression levels of 12 LibHLHs and those of the three structural genes. Notably, only LibHLH25 is part of the subgroup IIIf, which has been found to be associated with the regulation of the flavonoid biosynthesis pathway. However, other IIIf subgroup members, such as LibHLH50, LibHLH57, LibHLH61, and LibHLH62, displayed distinct expression patterns compared to LibHLH25, indicating potential functional diversification within this subgroup. Structural differentiation is a common feature among duplicate genes, often resulting in paralogous genes with functional distinctions [48]. The LibHLHs that belong to subgroup IIIf genes exhibit variations in exon-intron organization, which may contribute to spatio-temporal differences in their functions. The remaining 11 genes are non-IIIf subgroup LibHLHs, with some subgroups linked to anthocyanin synthesis. For instance, the light-induced subgroup IIIb member bHLH64 in pears enhances anthocyanin biosynthetic gene expression by forming a complex with the MYB10 protein [57]. In L. indica, subgroup IIIb members LibHLH68, LibHLH106, and LibHLH142, homologous to ICE1 of A. thaliana, are involved in flavonoid accumulation during cold acclimation. Additionally, LibHLH112 of subgroup VIIa, homologous to PIF3, is pivotal in light-induced anthocyanin synthesis under far-red light [58,59].

5. Conclusions

The present study, based on the genome of L. indica, identified and characterized 150 LibHLH transcription factor genes. Bioinformatic analyses revealed the complexity and diversity of the LibHLH proteins in terms of structural features, gene organization, and cis-regulatory elements, underscoring their pivotal roles in plant growth and development, as well as responses to biotic and abiotic stresses. Expression profiling indicated that 12 LibHLH genes were differentially expressed in L. indica cultivars with distinct leaf coloration, and their expression patterns strongly correlated with three structural genes involved in anthocyanin biosynthesis. Notably, LibHLH25, belonging to the subgroup IIIf associated with flavonoid synthesis, and LibHLH68, LibHLH106, LibHLH142, and LibHLH112 may contribute to the temperature- and light-regulated anthocyanin accumulation in L. indica.

Supplementary Materials

The following supporting information can be downloaded at: https://www.mdpi.com/article/10.3390/horticulturae11101219/s1, Figure S1: Predicted cis-elements in the promoters of 150 LibHLHs. Table S1: Specific primer sequences used for qRT-PCR analysis. Table S2: 150 LibHLH genes information in this study. Table S3: Prediction of cis-elements in LibHLH promoters. Table S4: List of LibHLH genes with collinearity.

Author Contributions

Conceptualization, J.S. and L.F.; methodology, X.H.; software, Y.G.; validation, X.H. and A.D.; formal analysis, L.F. and Y.G.; writing—original draft preparation, L.F. and Y.G.; writing—review and editing, L.F.; visualization, A.D.; project administration, J.S. and L.F.; funding acquisition, J.S. All authors have read and agreed to the published version of the manuscript.

Funding

This research was supported by the High Level Talent Research Start-Up Fund Project of Shandong Agriculture and Engineering University (No. BSQJ202304) and Sanming Projects Construction Project: “Famous Discipline”—Horticulture of Shandong Agriculture and Engineering University.

Data Availability Statement

The original contributions presented in this study are included in the article/supplementary material. Further inquiries can be directed to the corresponding author.

Conflicts of Interest

The authors declare no conflicts of interest.

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Figure 1. Phylogenetic analysis of the bHLH gene family in L. indica. The phylogenetic tree was constructed based on the representative protein sequences of 150 bHLH genes from L. indica and 70 bHLH genes from A. thaliana. The names of different subfamilies were designated using Greek numerals and distinguished by colors. All AtbHLHs were marked in red font.
Figure 1. Phylogenetic analysis of the bHLH gene family in L. indica. The phylogenetic tree was constructed based on the representative protein sequences of 150 bHLH genes from L. indica and 70 bHLH genes from A. thaliana. The names of different subfamilies were designated using Greek numerals and distinguished by colors. All AtbHLHs were marked in red font.
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Figure 2. Analysis of protein motifs, conserved domains, and gene structures of LibHLHs. (A) Phylogenetic classification of the LibHLH gene family. (B) Composition of conserved motifs in the LibHLH gene family, with varied colors denoting different motifs. (C) Types and distributions of conserved domains in the LibHLH gene family, with distinct colors denoting different conserved domains. (D) Exon—intron structures of the LibHLH gene family.
Figure 2. Analysis of protein motifs, conserved domains, and gene structures of LibHLHs. (A) Phylogenetic classification of the LibHLH gene family. (B) Composition of conserved motifs in the LibHLH gene family, with varied colors denoting different motifs. (C) Types and distributions of conserved domains in the LibHLH gene family, with distinct colors denoting different conserved domains. (D) Exon—intron structures of the LibHLH gene family.
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Figure 3. Location of LibHLHs on the chromosomes of L. indica. The scale bar represents the genome size (Mb). The distribution of each LibHLH gene is marked with a black line on the band. The brown arcs behind some LibHLH genes indicate that these gene pairs are tandem-repeated gene pairs.
Figure 3. Location of LibHLHs on the chromosomes of L. indica. The scale bar represents the genome size (Mb). The distribution of each LibHLH gene is marked with a black line on the band. The brown arcs behind some LibHLH genes indicate that these gene pairs are tandem-repeated gene pairs.
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Figure 4. Collinearity analysis of LibHLHs. Red lines indicate LibHLHs with collinear relationships. The blue area represents a high gene density, while the red area shows a low gene density.
Figure 4. Collinearity analysis of LibHLHs. Red lines indicate LibHLHs with collinear relationships. The blue area represents a high gene density, while the red area shows a low gene density.
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Figure 5. Collinearity analysis of LibHLHs among L. indica, A. thaliana, and P. granatum. Red lines represent LibHLHs with collinear relationships between L. indica and A. thaliana. Green lines represent LibHLHs with collinear relationships between L. indica and P. granatum.
Figure 5. Collinearity analysis of LibHLHs among L. indica, A. thaliana, and P. granatum. Red lines represent LibHLHs with collinear relationships between L. indica and A. thaliana. Green lines represent LibHLHs with collinear relationships between L. indica and P. granatum.
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Figure 6. The interaction network of LibHLHs, based on orthologues in A. thaliana. Nodes denote proteins, while edges signify the interaction relationships between proteins. Different line colors differentiate interaction types (Refer to the annotations on the STRING (https://cn.string-db.org/, accessed on 28 April 2025) for details). Parenthesized numbers (LibHLH) correspond to homologous genes in L.indica.
Figure 6. The interaction network of LibHLHs, based on orthologues in A. thaliana. Nodes denote proteins, while edges signify the interaction relationships between proteins. Different line colors differentiate interaction types (Refer to the annotations on the STRING (https://cn.string-db.org/, accessed on 28 April 2025) for details). Parenthesized numbers (LibHLH) correspond to homologous genes in L.indica.
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Figure 7. Analysis of the expression patterns of LibHLHs in the leaves of ‘Arapahoe’ (G) and ‘Ebony Embers’ (P). (A) Cluster analysis of LibHLHs expression profiles in the leaves of two crape myrtle cultivars at three developmental stages. (B) Leaf samples of L. indica ‘Ebony Embers’ (P) and ‘Arapahoe’ (G) at 5 days (S1), 15 days (S2), and 30 days (S3) after leaf germination. (C) Correlation analysis between LibHLH genes and anthocyanin structural genes LiCHS, LiANS, and LiDFR. The color gradient from red to green represents the correlation coefficient from high to low.
Figure 7. Analysis of the expression patterns of LibHLHs in the leaves of ‘Arapahoe’ (G) and ‘Ebony Embers’ (P). (A) Cluster analysis of LibHLHs expression profiles in the leaves of two crape myrtle cultivars at three developmental stages. (B) Leaf samples of L. indica ‘Ebony Embers’ (P) and ‘Arapahoe’ (G) at 5 days (S1), 15 days (S2), and 30 days (S3) after leaf germination. (C) Correlation analysis between LibHLH genes and anthocyanin structural genes LiCHS, LiANS, and LiDFR. The color gradient from red to green represents the correlation coefficient from high to low.
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Figure 8. qRT-PCR analysis of 12 LibHLH genes in leaves at different developmental stages of two cultivars. The value is the mean ± SD of the biological duplicate samples.
Figure 8. qRT-PCR analysis of 12 LibHLH genes in leaves at different developmental stages of two cultivars. The value is the mean ± SD of the biological duplicate samples.
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Feng, L.; Guo, Y.; Han, X.; Ding, A.; Shu, J. Genome-Wide Identification of the bHLH Gene Family and Expression Analysis in Anthocyanin Synthesis in Lagerstroemia indica Leaves. Horticulturae 2025, 11, 1219. https://doi.org/10.3390/horticulturae11101219

AMA Style

Feng L, Guo Y, Han X, Ding A, Shu J. Genome-Wide Identification of the bHLH Gene Family and Expression Analysis in Anthocyanin Synthesis in Lagerstroemia indica Leaves. Horticulturae. 2025; 11(10):1219. https://doi.org/10.3390/horticulturae11101219

Chicago/Turabian Style

Feng, Lu, Yanhong Guo, Xu Han, Aiqin Ding, and Jing Shu. 2025. "Genome-Wide Identification of the bHLH Gene Family and Expression Analysis in Anthocyanin Synthesis in Lagerstroemia indica Leaves" Horticulturae 11, no. 10: 1219. https://doi.org/10.3390/horticulturae11101219

APA Style

Feng, L., Guo, Y., Han, X., Ding, A., & Shu, J. (2025). Genome-Wide Identification of the bHLH Gene Family and Expression Analysis in Anthocyanin Synthesis in Lagerstroemia indica Leaves. Horticulturae, 11(10), 1219. https://doi.org/10.3390/horticulturae11101219

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