Genome-wide identification of basic helix-loop-helix (bHLH) genes in Pinellia ternata and their responses to abiotic stress and hormone treatments

Abstract

Background

Basic helix-loop-helix (bHLH) transcription factors constitute the second-largest transcription factor family in plants. They are widely distributed across plant genomes and play vital regulatory roles in plant growth and development, secondary metabolism, and responses to abiotic stress. Although the bHLH gene family has been widely identified and analyzed in many plant species, systematic genome-wide identification and functional analysis of bHLH genes in Pinellia ternata are still lacking.

Results

In this study, a total of 140 PtbHLH genes were identified, of which 135 were unevenly distributed on 13 chromosomes. Phylogenetic analysis with Arabidopsis thaliana divided the PtbHLH genes into 24 subfamilies, with no members clustered in subfamily VI. Gene structure and conserved motif analysis showed that members of the same subfamily had highly similar structural characteristics. Collinearity analysis identified three paralogous pairs among the 140 PtbHLH genes. Cross-species collinearity analysis revealed eight syntenic pairs between P. ternata and A. thaliana, 76 between P. ternata and Nicotiana tabacum, and 50 between P. ternata and Oryza sativa. Cis-element prediction showed that all PtbHLH promoters contained hormone-responsive elements, stress-responsive elements and transcription factor binding sites. Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) enrichment indicated that PtbHLH genes were mainly involved in hormone signal transduction, environmental signal response and transcriptional regulation. Protein–protein interaction (PPI) analysis suggested that PtbHLH68 was predicted as a putative interaction hub interacting with multiple family members. Tissue expression profiles showed that PtbHLH genes had diverse expression patterns, with most highly expressed in roots and tubers. Under high-temperature and drought stress, PtbHLH9 was most sensitive to high temperature, and PtbHLH85 was most sensitive to drought. Under methyl jasmonate (MeJA), abscisic acid (ABA) and salicylic acid (SA) treatments, PtbHLH73 responded most strongly to MeJA, PtbHLH7 to ABA, and PtbHLH48 responded significantly to all three hormones. These four genes serve as core candidates for exploring PtbHLH functions in abiotic stress tolerance and hormone signal transduction.

Conclusion

This study provides a solid foundation for understanding the functional mechanism of PtbHLH transcription factors in Pinellia ternata, and offers a valuable reference for the further screening of PtbHLH genes related to abiotic stress tolerance and hormone signaling pathways.

Introduction

Basic helix-loop-helix (bHLH) transcription factors contain a conserved basic DNA-binding domain and a helix-loop-helix (HLH) dimerization domain. As the second-largest transcription factor superfamily in plants, bHLH proteins are widely distributed in plant genomes and play vital regulatory roles in plant growth and development, secondary metabolism, and abiotic stress responses (Ledent & Vervoort, 2001; Lei et al., 2024; Song et al., 2025). Heim et al. (2003) first classified 133 Arabidopsis thaliana bHLH proteins into 12 subfamilies based on structural features to facilitate functional prediction. In terms of growth and development, SlMYB72 and SlMYB75 antagonistically regulate tomato trichome formation and SlCycB2 expression via the MYB-bHLH-WD40 complex (Qi et al., 2025); wheat TaPGS1, specifically expressed in grains 5–20 days post-anthesis, enhances grain weight/size and nutrient contents by activating TaFl3 (Guo et al., 2022); maize ZmBES1/BZR1-5 and ZmBES1/BZR1-4 positively modulate grain development and yield (Feng et al., 2025). For secondary metabolism, Betula platyphylla BpbHLH9 promotes triterpenoid accumulation by upregulating related biosynthetic genes (Yin et al., 2017); ABA/JA-induced Artemisia annua AabHLH113 boosts artemisinin and dihydroartemisinic acid levels via targeting DBR2 and ALDH1 (Yuan et al., 2023); Salvia miltiorrhiza SmbHLH148 activates phenolic acid and tanshinone biosynthesis through ABA/MeJA signaling (Xing et al., 2018). In abiotic stress responses, soybean GmPIF4b alleviates heat damage and preserves reproductive organ integrity under high temperatures (Arya, Singh & Bhalla, 2024); potato StbHLH47 negatively regulates drought tolerance (Wang et al., 2025); SA enhances Cinnamomum camphora drought resistance by inducing stress-related genes and activating bHLH/MYB-mediated hormone signaling (Zhao et al., 2025). With maturing analysis technologies, genome-wide identification of transcription factor families has become a standardized bioinformatic workflow, and bHLH families have been identified in diverse plants, with member counts varying across species—164 in Carya illinoinensis (Zou et al., 2025), 122 in Andrographis paniculata (Xu et al., 2022), 83 in Suaeda aralocaspica (Wei, Cao & Lan, 2022), 12 in Vitis vinifera (Sun et al., 2025a), and 124 in Chinese chestnut (Li et al., 2025a)—laying a foundation for subsequent structural and functional studies of these genes.

Pinellia ternata (Thunb.) Breit., a perennial herb belonging to the Pinellia genus of the Araceae family, has a long history of medicinal use as its dried tuber, which is pungent, warm, and slightly toxic in nature, and acts on the lung, spleen, and stomach meridians with traditional efficacy in resolving dampness and phlegm, relieving vomiting, and dissipating abdominal masses (Liang et al., 2025). As a widely used bulk medicinal herb, the growth and development of P. ternata and the content of pharmacodynamic metabolites have attracted substantial attention. P. ternata mainly relies on asexual reproduction, which is closely associated with the formation and abscission of bulbils; high levels of jasmonic acid (JA), ABA, isopentenyladenosine (IPA), and SA have been detected in bulbils during their initial growth from petioles (Mou et al., 2024). During different growth and developmental stages of P. ternata corms, the contents of JA and ABA generally show an upward trend, which may enhance the corms’ adaptability to the environment during the transition from tender to mature tissues (Jia et al., 2025). P. ternata prefers shaded and humid growth conditions; sustained ambient temperatures above 35 °C or water-deficit (drought) stress will accelerate the senescence and death of its above-ground parts, leading to the “seedling lodging” phenomenon (Xue et al., 2019). High temperature and drought can cause damage to P. ternata petioles and leaves, resulting in abnormal physiological indices such as a significant increase in malondialdehyde (MDA) content (Liu et al., 2024), and studies have indicated that seedling lodging in P. ternata is essentially a form of programmed cell death. Alkaloids and nucleosides are the main bioactive components responsible for the medicinal effects of P. ternata (Chen et al., 2023); Duan et al. (2019) found that SA treatment of in vitro petiole culture systems for P. ternata microtuber induction significantly increased the contents of total alkaloids as well as guanosine, inosine, and ephedrine. In summary, high temperature, drought, and hormone treatments all exert notable impacts on the growth, development, and metabolism of P. ternata.

Despite widespread characterization of bHLH families across many plant taxa, no systematic research targeting the bHLH gene family has been reported for the medicinal herb P. ternata so far. Routine genome-wide identification pipelines alone cannot represent the innovation of this study; instead, the core value lies in the first comprehensive dissection of the bHLH repertoire specific to P. ternata. Using the publicly available P. ternata whole-genome assembly (Xue et al., 2024), we identified 140 PtbHLH family members and carried out multi-layered analyses covering physicochemical properties, gene and protein conserved architectures, phylogenetic relationships, intra- and interspecific collinearity, cis-acting regulatory elements, GO and KEGG functional annotations, and protein-protein interactions of these PtbHLH genes. Moreover, we analyzed the tissue-specific expression profiles of PtbHLH genes using public transcriptome datasets and selected representative genes to clarify their expression patterns under drought, high-temperature stress, and exogenous hormone treatments. This work provides a species-specific baseline for further functional exploration of bHLH regulators governing stress adaptation and medicinal metabolite biosynthesis in P. ternata.

Materials and Methods

Identification of PtbHLH gene family in P. ternata

Whole-genome sequence data of P. ternata with genome assembly Project ID PRJCA016404 and its corresponding gene annotation file were retrieved from the National Genomics Data Center (NGDC, https://ngdc.cncb.ac.cn/). A. thaliana bHLH protein reference sequences were downloaded from TAIR (https://www.arabidopsis.org). Two strategies were combined to capture candidate PtbHLH proteins: (1) BLASTp alignment against Arabidopsis bHLH sequences with E-value cutoff = 1e−5; (2) HMMER v3.0 screening using the bHLH hidden Markov model PF00010 from Pfam, with default gathering threshold as cutoff and a minimum conserved domain coverage of 70% retained. All candidate proteins were further verified for intact bHLH conserved domains via SMART (http://smart.embl.de) and NCBI CDD (https://www.ncbi.nlm.nih.gov/cdd/). Redundant protein sequences with 100% amino acid identity were removed using TBtools, and sequences lacking complete bHLH domains were eliminated to obtain the final set of 140 non-redundant PtbHLH proteins. TBtools was applied to predict molecular weight, theoretical isoelectric point and amino acid length of each PtbHLH protein (Chen et al., 2020), and subcellular localization was predicted via DeepLoc-2.1 (https://services.healthtech.dtu.dk/services/DeepLoc-2.1/).

Phylogenetic analysis and chromosomal location

Conserved amino acid sequences of bHLH proteins from P. ternata and A. thaliana were subjected to alignment using the Muscle algorithm implemented in MEGA11. A Maximum Likelihood (ML) phylogenetic tree was constructed with 1,000 bootstrap replicates, which was subsequently visualized and refined via the online tool ChiPlot (https://www.chiplot.online). The PtbHLH family was classified with reference to the established A. thaliana bHLH classification system from prior studies. TBtools was used to extract chromosome length and positional information of PtbHLH members from the P. ternata genome sequence and GFF file (Chen et al., 2020).

Motif, gene structure analysis of PtbHLHs

PtbHLH protein sequences were uploaded to the MEME server (https://meme-suite.org/meme/tools/meme) for conserved motif analysis, with the maximum number of motifs set to 10. TBtools was employed to visualize the genomic GFF3 file of P. ternata and the predicted conserved motifs.

Cis-element analysis of PtbHLHs

Promoter sequences (2,000 bp upstream of PtbHLH genes) were retrieved with TBtools software, and cis-acting element prediction for these promoter sequences was performed using the PlantCARE database (http://bioinformatics.psb.ugent.be/webtools/plantcare/html/).

Collinearity analysis between and within species

Genome data of three plant species (A. thaliana, N. tabacum, and O. sativa) were downloaded from Ensembl Plants (http://plants.ensembl.org/index.html). The One Step MCScanX-Super Fast module in TBtools was used to analyze the collinearity relationships within the P. ternata genome and between P. ternata and the three aforementioned species, with the analysis results visualized via TBtools (Chen et al., 2020).

GO, KEGG, and interaction network analysis

GO and KEGG enrichment analyses of PtbHLH protein sequences were performed using TBtools, with the results visualized (Chen et al., 2020). Only the 140 PtbHLH proteins were set as the foreground gene set for enrichment, and the full P. ternata transcriptome was not adopted as the background reference. PPI network analysis was performed using the STRING database (https://cn.string-db.org/).

Expression analysis of PtbHLHs

Published P. ternata RNA-seq data was downloaded from the NGDC website (https://ngdc.cncb.ac.cn/, accession number PRJCA016404) (Xue et al., 2024), covering different tissues (leaves, petioles, flowers, tubers, roots). Expression levels (transcripts per million, TPM) were calculated, and the results were visualized as heatmaps using the pheatmap package. One-month-old P. ternata plants were exogenously treated with 100 μM MeJA, 100 μM ABA, and 25 μM SA for 1 h, followed by transcriptome sequencing (NGDC accession number PRJCA059529) with three biological replicates per treatment. All RNA-seq data generated in this study are publicly available at NGDC under accession PRJCA059529. The statistical power of the hormone-treated transcriptome experiment was calculated using an RNA-seq power analysis tool. Parameters were set as follows: three biological replicates, average sequencing depth of 100 reads per gene, coefficient of variation (CV) of 0.4, fold change of two, and significance level α = 0.05, the calculated power was 0.48. TPM values under different hormone treatments were visualized using TBtools (Chen et al., 2020).

Plant material and treatments

Uniform-sized P. ternata tubers were planted in the culture room of the Shaanxi Provincial and Ministerial Co-construction Center for Innovation in Chinese Medicinal Resources Industrialization, under controlled conditions of 25 °C and a 12 h/12 h light/dark cycle. For abiotic stress and hormone induction experiments, 1-month-old plants with three leaves were treated with 42 °C (high-temperature stress, short-term treatment without triggering seedling lodging and programmed cell death), 15% polyethylene glycol 6000 (PEG 6000) for drought simulation (seedlings only showed mild leaf curling within 24 h of treatment and could fully recover after rehydration, without severe lethal leaf necrosis), 100 μM MeJA, 100 μM ABA, and 25 μM SA, respectively. Leaves were harvested at 0, 1, 3, 6, 12, and 24 h post-treatment, promptly snap-frozen in liquid nitrogen, and stored at −80 °C for subsequent use. Each single biological replicate contained three pooled P. ternata plants, and the entire experiment was conducted with three independent biological replicates (nine individual plants in total per sampling time point); samples collected at 0 h served as untreated control groups. This experimental setup guaranteed reliable biological reproducibility.

RNA isolation and quantitative reverse transcription polymerase chain reaction validation

Total RNA was isolated from various tissues of P. ternata using the TIANGEN (Beijing, China) Polysaccharide & Polyphenol Total RNA Extraction Kit (Catalog No. DP441). The isolated RNA was reverse-transcribed into cDNA in accordance with the manufacturer’s instructions for the TIANGEN (Beijing, China) Reverse Transcription Premix Kit (Catalog No. KR118). Specific primers for quantitative reverse transcription polymerase chain reaction (qRT-PCR) were designed using Primer 5 software based on the CDS sequences of 11 selected PtbHLH genes and synthesized by Sangon Biotech (Shanghai, China) (Table S1). With cDNA as the template and the 18S gene as the internal reference gene (Xue et al., 2019), qRT-PCR was performed using SuperReal PreMix Plus (SYBR Green, Catalog No. FP205-02) on the Analytik Jena qTOWER system. The PCR mixture consisted of 1 μL cDNA, 10 μL 2× SuperReal PreMix Plus, 0.6 μL of each primer, and 7.8 μL ddH₂O. The qRT-PCR program included pre-denaturation at 95 °C for 15 s, followed by 40 cycles of denaturation at 95 °C for 10 s and annealing/extension at 60 °C for 32 s. After amplification, a melting curve procedure was performed: temperature was increased from 60 °C to 95 °C at a ramp rate of 0.5 °C/s, and fluorescence signals were continuously acquired during the temperature rise to verify specific amplification of target fragments. Each PtbHLH gene was detected based on three independent biological replicate samples. Relative gene expression levels were calculated using the 2^−ΔΔCt method (Livak & Schmittgen, 2001; Li et al., 2025b), and data were analyzed and visualized with GraphPad (Boston, MA, USA) Prism (v. 10.4.1).

Results

Identification of bHLH genes in P. ternata genome

A total of 140 bHLH gene sequences were identified through genome-wide analysis of P. ternata and named PtbHLH1-PtbHLH140 sequentially (Table S2). The number of amino acids encoded by genes in this family ranged from 98 to 794 aa, with an average of 397 aa. The relative molecular weight varied from 11,367.59 to 88,279.59 Da, with a mean value of 43,034.07 Da. The theoretical isoelectric point (pI) spanned from 4.74 to 10.74, with an average of 6.60. Among these proteins, 96 had a pI below 7, accounting for 68.6% of the total and classifying them as acidic proteins. Only five members, PtbHLH1/14/16/98/118, had an instability index below 40, indicating that they are stable proteins. The aliphatic index ranged from 48.69 to 100.47, suggesting significant differences in thermal stability among family proteins. The grand average of hydropathicity (GRAVY) values were between −1.261 and 0.196, and all proteins were hydrophilic except PtbHLH19. Subcellular localization prediction showed that all PtbHLH proteins were localized in the nucleus, with PtbHLH100 additionally localized in the cytoplasm.

Phylogenetic analysis and chromosome location of the PtbHLH family

A ML phylogenetic tree was constructed using conserved amino acid sequences of 140 P. ternata bHLH proteins and 158 A. thaliana bHLH proteins (Fig. 1). Phylogenetic analysis clustered the total 298 bHLH protein sequences of two species into 25 subfamilies, among which 24 subfamilies contained PtbHLH genes and subfamily VI had no P. ternata bHLH members. Based on the phylogenetic tree, the 24 subfamilies containing PtbHLH genes were further grouped into 11 major clades. For qRT-PCR validation, one representative gene was selected from each of these 11 major clades, covering the full phylogenetic diversity of the family. Statistical analysis of the number of bHLH genes from P. ternata and A. thaliana in each subfamily was performed (Table S3). The results showed that no PtbHLH genes were detected in subfamily VI, implying potential gene loss or functional divergence during the evolution of P. ternata and subfamily XII contained the largest number of PtbHLH genes (21 in total). The number of PtbHLH genes in subfamilies IIIb, IIIc, IIId, IVd, Vb, VIIb, and XII was significantly higher than that of AtbHLH genes (ratio ≥1.50), implying that these subfamilies have undergone expansion during the evolution of P. ternata and may be associated with specific functional adaptations. In contrast, the gene number ratios of subfamilies Ib, IIIf, IX, VIIIa, and XI were ≤0.50, indicating a marked reduction in PtbHLH gene count in these subfamilies, which may result from gene loss or functional differentiation. For subfamilies Ia, IIIa, IIIe, and X, the gene number ratios were close to 1.00, suggesting that the gene numbers of these subfamilies have changed little between the two species and their functions may be more conserved. The 140 PtbHLH genes exhibited non-uniform distribution across 13 complete chromosomes and five unanchored scaffold fragments (Fig. 2). Among the 13 chromosomes, Chr09 harbored the most PtbHLH genes (20), followed by Chr02 (17), Chr10 (16), and Chr05 (15). In contrast, Chr12 (1) and Chr11 (3) contained the fewest genes. The gene numbers on the remaining chromosomes were intermediate: Chr01 (14), Chr03 (10), Chr04/Chr08 (6 each), Chr06 (12), Chr07 (11), and Chr13 (4).

Figure 1. Phylogenetic tree of 140 PtbHLH and 158 AtbHLH proteins.

Different background colors represent distinct subfamilies.

Figure 2. Chromosomal localization of PtbHLH genes.

Analysis of gene structure, motif and conserved domains of PtbHLHs

To gain deeper insights into the sequence and structural characteristics of the PtbHLH gene family, a phylogenetic tree based on PtbHLH protein sequences was generated with MEGA11 (Fig. 3A). A motif distribution map was constructed through the MEME database (Fig. 3B), while detailed information regarding the composition of each motif is presented in Fig. S1. A total of 10 conserved motifs were identified, and the motif count in the 140 PtbHLH proteins varied from 1 to 10. Among them, motif 1 and motif 2 were the two most conserved motifs, widely distributed across PtbHLH family members and closely positioned in sequences (Fig. 3C). Conserved domain analysis via the SMART database revealed that all 140 PtbHLH proteins contained either the bHLH_AT or bHLH_SF domain (Fig. 3D). Gene structure analysis showed that some PtbHLH genes possessed multiple untranslated regions (UTRs), while others lacked these elements. The number of introns in PtbHLH genes varied dramatically, ranging from 0 to 9 (Fig. 3D). Generally, members within the same subfamily exhibited similar or identical gene structures, whereas significant differences were observed between different subfamilies.

Figure 3. Phylogenetic and gene structure analysis of PtbHLH genes.

(A) Phylogenetic tree of PtbHLH proteins. (B) Conserved motif analysis of PtbHLH proteins, with a total of 10 predicted motifs represented by different colored boxes. (C) Domain pattern analysis of PtbHLH proteins. (D) Gene structure analysis of PtbHLH genes.

Cis-acting element analysis of the promoter region of PtbHLHs

Analysis of cis-acting elements in the promoter regions of 140 PtbHLH genes in P. ternata (Fig. 4) showed that these elements could be classified into three categories: growth and development-related, hormone-responsive, and abiotic stress and defense-responsive elements. Among growth and development-related elements, light-responsive elements (L) were detected in all PtbHLH genes and highly abundant in most of them (e.g., the L value of PtbHLH25 and PtbHLH129 reached 37), which are common motifs widely existing in plant promoters. Tissue- or process-specific elements such as cell cycle (Cyc), circadian rhythm (Circ), seed-specific (Sd), and root-specific (Rt) elements were only detected in a small number of genes (PtbHLH11/14 contained Cyc elements; PtbHLH14/16 contained Sd elements). Meanwhile, metabolism-related elements including MBSI (MYB-binding site involved in flavonoid biosynthesis) and Zein (zein metabolism-related) were also identified in partial members. For hormone-responsive elements, abscisic acid (ABRE), salicylic acid (SA), and methyl jasmonate (MeJA) responsive elements were widely distributed (e.g., the SA value of PtbHLH25 reached 17; the MeJA value of PtbHLH26 and PtbHLH33 was 5), while auxin (Aux) and gibberellin (GA) responsive elements were relatively rare. In the category of abiotic stress and defense-responsive elements, salt stress-responsive elements (St) were highly enriched in some genes (the St value of PtbHLH112 was 20; that of PtbHLH1/22 was 16). Low-temperature-responsive (LT), anaerobic induction (Ana), and anoxic enhancement (Anx) elements were only present in a few genes as required. Additionally, MBS (MYB-binding site involved in drought inducibility) and Eli (elicitor activation-related) elements showed member-specific distribution patterns. Overall, the element composition of the PtbHLH gene family displayed characteristics of conserved core elements and differentiated specific elements, which only provides predictive clues for potential functional divergence rather than verifying that all PtbHLH members participate in hormone and stress signaling pathways. These cis-element features may be linked to the regulation of P. ternata growth and development, hormone signal transduction, and stress adaptation, offering candidate directions for subsequent gene function verification. Only qualitative classification and counting of cis-elements were performed here, without statistical functional annotation such as hypergeometric test against random promoter background sequences.

Figure 4. Analysis of cis-acting elements in the bHLH gene family of P. ternata.

Numbers in grid cells represent the number of cis-acting elements.

Gene duplication, and collinearity analysis of PtbHLHs

Three segmental duplication events were detected in the PtbHLH gene family (Fig. 5A, Table 1), involving the gene pairs PtbHLH88/80, PtbHLH87/130, and PtbHLH64/2. Notably, the Ks value of PtbHLH87/PtbHLH130 reached 1.1736, implying that this pair derives from an ancient duplication with saturated synonymous substitutions. All these gene pairs exhibited Ka/Ks (non-synonymous substitution rate/synonymous substitution rate) <1, suggesting that the PtbHLH gene family has experienced purifying selection throughout its evolutionary process. Only one tandem duplicated pair (PtbHLH28 and PtbHLH29) was screened out with criteria of interval <100 kb and protein identity >70%. To investigate the evolutionary relationships between bHLH genes of P. ternata and those from other plant species, interspecific collinearity analysis was conducted among P. ternata, A. thaliana, N. tabacum, and O. sativa. The results (Fig. 5B) showed that there were 8 collinear gene pairs between P. ternata and A. thaliana, 76 collinear gene pairs between P. ternata and N. tabacum, and 50 collinear gene pairs between P. ternata and O. sativa. These findings suggest that the bHLH gene family of P. ternata shares a closer homologous evolutionary relationship with that of N. tabacum.

Figure 5. Collinearity analysis of P. ternata bHLH genes.

(A) From outside to inside: chromosomes, gene density on chromosomes. (B) Collinearity between A. thaliana, N. tabacum, O. sativa and P. ternata (focusing on bHLH genes).

Table 1. Ka/Ks values of duplicated gene pairs in P. ternata.

GeneName1	GeneName2	Ka	Ks	Ka/Ks
PtbHLH88	PtbHLH80	0.2324	0.5438	0.4274
PtbHLH87	PtbHLH130	0.1840	1.1736	0.1568
PtbHLH64	PtbHLH2	0.0183	0.0395	0.4644

GO, KEGG enrichment and protein-protein interaction analysis of PtbHLHs

GO enrichment was performed solely on the 140 PtbHLH family proteins, without the whole P. ternata transcriptome as background. GO functional annotation of the PtbHLH gene family in P. ternata (Fig. 6A) revealed the following key findings: In terms of molecular function, DNA-binding transcription factor activity, core promoter sequence-specific DNA binding, and DNA binding were the most significant terms. A total of 84 target genes were annotated to transcription factor activity-related terms, reflecting the inherent conserved characteristic of bHLH transcription factors in regulating gene expression. For cellular component, the nucleus was the major enriched localization, with 68 genes localized therein—consistent with the subcellular localization requirement for transcriptional regulation. For biological process, the genes were significantly enriched in nucleic acid metabolism, RNA metabolism, and pathways associated with transcriptional regulation. KEGG enrichment results (Fig. 6B) indicated that the genes were mainly concentrated in pathways such as signal transduction, plant hormone signal transduction, transcription factors, plant MAPK signaling pathway, and plant circadian rhythm. This further suggests that PtbHLH genes may modulate the growth and development, stress adaptation, and secondary metabolism of P. ternata via hormone signal transduction, environmental signal response, and transcription regulation, offering potential clues for follow-up functional research.

Figure 6. GO, KEGG enrichment and regulatory network analysis of PtbHLH genes.

(A) GO functional annotation of PtbHLH genes. (B) KEGG functional annotation of PtbHLH genes. (C) PtbHLH protein-protein interaction analysis based on A. thaliana homologous genes. Among the lines in (C): purple lines represent experimentally determined interactions; blue lines represent interactions from curated databases; yellow lines represent text-mining evidence; black lines represent co-expression relationships; light purple lines represent interactions inferred from protein homology.

Screening and interaction prediction of 140 bHLH gene sequences in P. ternata were conducted using two criteria: sequence identity with A. thaliana bHLH proteins >40% (homology standard) andinteraction score >0.7 (credibility threshold). Finally, 27 PtbHLH members with mutual regulatory relationships were obtained (Fig. 6C). These members formed a modular protein interaction network: for example, PtbHLH17/1/20/68/133/122/124/59/27, and PtbHLH135/65/84/85 respectively formed multiple dense interaction clusters. Some members (e.g., PtbHLH68) served as “core nodes” interacting with multiple members, which are predicted to be key targets of the regulatory mechanism of this gene family. It should be noted that the 40% identity cutoff may lead to false-positive interactions, and all predicted protein interactions need further experimental verification such as Y2H or Co-IP in subsequent work.

Expression pattern of PtbHLHs in different tissues and hormone treatments of P. ternata

Members of the PtbHLH family exhibited divergent tissue-preferential expression across five tissues (leaf, petiole, flower, tuber, root) (Fig. 7A), and their expression preferences were closely linked to tissue identity. PtbHLH4, PtbHLH31 and PtbHLH104 displayed abundant transcript accumulation in leaves; PtbHLH26, PtbHLH57 and PtbHLH120 were highly transcribed in petioles. PtbHLH35 showed the highest expression level in flowers, accompanied by moderate expression of PtbHLH21, PtbHLH77 and PtbHLH50, while PtbHLH100 was exclusively detected in flowers as a flower-preferential gene. PtbHLH2, PtbHLH66 and PtbHLH125 represented the dominant highly expressed genes in tubers, and PtbHLH18, PtbHLH28 and PtbHLH139 showed relatively high transcript abundance in roots. A subset of genes displayed tissue-restricted transcriptional absence: PtbHLH1 was undetectable in flowers and roots, with only weak expression in leaves, petioles and tubers; PtbHLH6 had extremely low transcript levels solely in roots and was absent from all other tissues; PtbHLH71 maintained negligible or undetectable expression across all examined tissues. These distinct expression profiles reflect tissue-based functional differentiation within the PtbHLH family. Genes with TPM < 1 were regarded as transcriptionally undetectable in corresponding tissues.

Figure 7. Expression analysis of PtbHLH genes.

(A) Tissue-specific expression patterns of PtbHLH genes in five tissues (leaf, petiole, flower, tuber, root) of P. ternata. (B) Expression changes of PtbHLH genes in P. ternata leaves under different hormone treatments (100 μM MeJA, 100 μM ABA, 25 μM SA) for 1 h.

Transcriptome sequencing was conducted on 1-month-old P. ternata leaves harvested 1 h after exogenous treatment with 100 μM MeJA, 100 μM ABA and 25 μM SA (Fig. 7B). This dataset only captured short-term early transcriptional shifts of the 140 PtbHLH family members upon hormone treatment; full-transcriptome differential expression statistical testing was not performed, and the heatmap was generated purely based on extracted TPM values of bHLH genes. Under MeJA treatment, PtbHLH73, PtbHLH112, PtbHLH139, PtbHLH60 and PtbHLH85 presented clearly elevated TPM values, whereas PtbHLH51, PtbHLH89, PtbHLH66, PtbHLH68 and PtbHLH31 showed obvious reductions in transcript abundance. Following ABA treatment, PtbHLH7, PtbHLH85, PtbHLH64, PtbHLH22 and PtbHLH103 displayed increased TPM levels, while PtbHLH89, PtbHLH66, PtbHLH68, PtbHLH126 and PtbHLH46 exhibited reduced transcript accumulation. After SA treatment, elevated TPM signals were observed for PtbHLH67, PtbHLH77 and PtbHLH7, and decreased transcript levels were detected for PtbHLH115, PtbHLH24 and PtbHLH13. Overall, the magnitude of TPM variation triggered by 1 h SA exposure was weaker than the transcriptional fluctuations induced by MeJA and ABA. Several genes including PtbHLH64, PtbHLH22, PtbHLH85, PtbHLH89, PtbHLH66 and PtbHLH68 shared similar TPM alteration trends under short-term MeJA and ABA treatments. To characterize dynamic expression responses over extended timeframes, 11 representative genes from distinct evolutionary clades were selected for time-series qRT-PCR detection covering 0–24 h. The qRT-PCR results revealed delayed transcriptional responses at 6–24 h for multiple candidate genes; nevertheless, these quantitative expression patterns only apply to the 11 selected representatives and cannot be extrapolated to all members of the PtbHLH gene family.

Response of PtbHLHs to abiotic stress and hormone treatment

To investigate the response characteristics of PtbHLH genes to abiotic stresses and plant hormones, this study used 1-month-old P. ternata as materials, and conducted treatments including high temperature (42 °C), drought (15% PEG6000), 100 μM MeJA, 100 μM ABA, and 25 μM SA, to analyze the expression dynamics of 11 PtbHLH genes (one selected from each of the 11 major evolutionary clades) in leaves (Fig. 8). Under 42 °C high-temperature stress: PtbHLH9/27/48/65/67/127 showed no significant upregulation at 1 and 3 h of treatment, with their expression peaks delayed until 12 or 24 h; PtbHLH53 was significantly induced at 3 h; PtbHLH73/76/90 were significantly downregulated at 1 h, then gradually increased with prolonged treatment duration, reaching peaks at 12 or 24 h; PtbHLH85 had low expression at 1 and 3 h, significantly increased from 6 h onwards, and maintained high-level expression after 12 h. Notably, PtbHLH9 showed extremely significant response to 42 °C high temperature, with the expression increase amplitude at the peak stage significantly higher than that of some other members in the family, implying it may be a candidate gene associated with heat stress response. Under 15% PEG6000-simulated drought treatment: most genes generally showed expression inhibition at 1 and 3 h, their relative expression levels significantly increased at 6 and 12 h, and significantly decreased at 24 h; PtbHLH53 had the basal expression at 0 h and maintained low levels after treatment; PtbHLH76 had a relatively gentle response pattern with no significant fluctuations in expression; most genes including PtbHLH9/27/48 reached expression peaks at 12 h, suggesting that 12 h may be a key time point for drought response.

Figure 8. Expression patterns of 11 PtbHLH genes in P. ternata leaves under abiotic stress and hormone treatments.

The treatment methods are as follows: (A) High temperature (42 °C); (B) drought (15% PEG6000-simulated drought); (C) 100 μM MeJA; (D) 100 μM ABA; (E) 25 μM SA. Error bars indicate the ± SD of three independent experiments. One-way ANOVA was performed after Brown–Forsythe/Bartlett variance homogeneity tests; post-hoc comparisons used Tukey’s HSD test. Different lowercase letters (P < 0.05) denote significant differences.

Analysis of the responses to plant hormones MeJA, ABA, and SA showed that these three hormones may participate in regulating plant growth and development. Under MeJA treatment: PtbHLH9/53/127 reached expression peaks at 24 h; PtbHLH27/65/76 had the highest expression at 0 h untreated control, followed by a downward trend. It is worth noting that PtbHLH48 and PtbHLH73 were significantly induced at 1 h of MeJA treatment, and their expression levels continued to decrease significantly thereafter—among them, the expression level of PtbHLH48 increased to approximately 60-fold that at 0 h, which may suggest that this gene participates in MeJA signal transduction. Under ABA treatment: except for PtbHLH73, all other genes were rapidly upregulated to peaks at 1 or 3 h, then significantly downregulated with prolonged time; PtbHLH73 had high expression at 0 h, decreased sharply after 1 h and maintained low levels, while PtbHLH48 and PtbHLH127 were most significantly induced by ABA, with their expression levels reaching thousands of times that at 0 h. Under SA treatment: most genes including PtbHLH9/27/53 showed a trend of first gradually increasing, reaching peaks at 3 or 6 h, then gradually decreasing—among them, PtbHLH48 and PtbHLH127 had significant induction effects, with their expression levels reaching 150-fold that at 0 h at a later time point, which may reflect the functional differentiation of this gene family in hormone signal response.

Discussion

Genome-wide identification of transcription factor families has become a routine bioinformatic pipeline in plant genomics, and the novelty of the present study does not lie in this standard identification workflow itself. Instead, its core value lies in the first systematic characterization of the bHLH gene family in the medicinal herb P. ternata, a species lacking prior relevant reports. The bHLH transcription factor family is among the largest transcription factor families in plants. It plays crucial roles in regulating plant growth and development, as well as in responses to abiotic stresses (Heim et al., 2003). To date, bHLH gene families have been reported in plants such as A. thaliana, O. sativa (Li et al., 2006), N. tabacum (Bai et al., 2021), Panax ginseng (Zhu et al., 2020), Scutellaria baicalensis (Sun et al., 2025b), and Perilla frutescens (Chen et al., 2024); however, no such report has been published for the medicinal plant P. ternata. This study identified 140 members of the PtbHLH family in P. ternata. The number of bHLH family members varies among different plants, which may be related to genome duplication events, evolutionary difference, or genome size variations among plant species (Qiao et al., 2019)—for example, there are 41 bHLH family members in Carthamus tinctorius (Yingqi et al., 2019), while there are 448 bHLH family members in Phyllostachys edulis (Cheng et al., 2018). Among the PtbHLH genes in P. ternata, 135 are unevenly distributed on 13 chromosomes, and five genes are located on unanchored scaffold fragments. Genome sequencing results of P. ternata show that the total length of the 13 chromosomes accounts for 95.46% of the total genome sequence (Xue et al., 2024), which may be caused by incomplete genome assembly.

At present, the biological functions of most PtbHLH members remain unclear. Analysis of their gene structure, motifs, and conserved domains shows that members of the same subfamily have similar structures and conserved motifs, indicating that members of the same subfamily may have certain functional similarities. Different P. ternata bHLH genes contain different numbers of exons and introns; however, in subfamilies IIIe and VIIIb, none of the P. ternata bHLH genes have introns, which supports the certain accuracy of subfamily classification. Ten conserved motifs were identified in the 140 P. ternata bHLH protein sequences. Among them, conserved Motif 2 is present in all P. ternata bHLH protein sequences, suggesting that it plays an irreplaceable role in the function of P. ternata bHLH genes. Through phylogenetic analysis of A. thaliana bHLH proteins, the bHLH family was classified into 24 subfamilies. No P. ternata bHLH genes were found in subfamily VI, indicating that partial differentiation has occurred between the bHLH gene families of P. ternata and A. thaliana during long-term evolution. This phenomenon has also been observed in plants such as Atractylodes lancea (Zhang et al., 2025), Lilium bakerianum var. rubrum (Gu et al., 2025), and Zanthoxylum armatum (Yi et al., 2025).

Cis-acting elements in the promoter region play an important role in regulating gene function (Fan et al., 2025). The cis-acting elements in the promoter region of P. ternata bHLH genes are mainly classified into three categories: growth and development-related, hormone-responsive, and abiotic stress-responsive elements. Every PtbHLH gene contains light-responsive elements, indicating that PtbHLH genes are regulated by light signals and are of great significance for coordinating plant growth and development. Most PtbHLH genes contain MeJA, SA, and ABA responsive elements. MeJA has a significant inductive effect on the accumulation of various plant secondary metabolites and can also act synergistically with other hormones such as ABA. In Artemisia annua (sweet wormwood), AaMYC2 and AabHLH113 induced by JA and ABA signals, respectively, activate CYP71AV1, double bond reductase (DBR2), and AaALDH1, which positively regulate the biosynthesis of artemisinin (Yuan et al., 2023). In terms of abiotic stress, studies have found that the enhancement of plant drought resistance induced by bHLH transcription factors is generally related to the signal transduction of ABA and JA pathways and the scavenging of reactive oxygen species (ROS) (Li et al., 2007). In Triticum aestivum, over expression of TabHLH1 can alter growth characteristics such as stomatal movement and leaf water loss rate under drought stress, and improve drought adaptability through the ABA pathway. Based on this, it is speculated that under hormone induction (such as JA and ABA) and drought stress, PtbHLH genes homologous to AaMYC2 and TabHLH1 may have similar functions.

GO functional annotation showed that PtbHLH genes are mainly localized in the nucleus, have DNA-binding ability, and are involved in primary metabolism and macromolecular biosynthesis processes. KEGG functional annotation indicated that PtbHLH genes are mainly involved in environmental information processing and plant hormone signal transduction processes. These enrichment results confirm that as transcription factors, P. ternata PtbHLH genes exert their functions by regulating other genes when affected by the external environment or induced by hormones, thereby maintaining the normal growth and development of plants. Analysis of the interaction relationship of PtbHLH proteins showed that PtbHLH68 was predicted as a putative interaction hub and interacts with multiple members. It is predicted that this protein may be a key target of the regulatory mechanism of the PtbHLH family.

The tissue-specific expression patterns of PtbHLH family genes in P. ternata reflect the functional differentiation of this family formed during evolution. The five tissue types (leaf, petiole, flower, tuber, root) all have core highly expressed members, and their expression preferences are closely associated with the physiological functions of each tissue: highly expressed genes in leaves may be involved in the regulation of photosynthesis; PtbHLH100 (flower-specific) and highly expressed PtbHLH35/21 in flowers are likely to play key roles in reproductive development; while core genes in tubers and roots may mediate processes such as nutrient storage, absorption, and transport. Transcriptome sequencing was performed on P. ternata leaves after 1 h of MeJA, ABA, and SA treatments. In the MeJA treatment group, the expression levels of genes such as PtbHLH73/112/139/60 were significantly increased, among which PtbHLH73/112 are MYC2 homologous genes. MYC2 genes responsive to MeJA have been reported in multiple plant species (Li et al., 2024). In Taxus chinensis, the JA-responsive transcription factor TcMYC2a directly regulates the expression of ERF transcription factors such as TcERF15 to control TcTASY expression, thereby promoting paclitaxel biosynthesis (Zhang et al., 2018). In Aquilaria sinensis, AsMYC2 regulates the expression of ASS1 through the JA signaling pathway and participates in the regulation of sesquiterpene biosynthesis (Xu et al., 2017). In Catharanthus roseus, overexpression of CrMYC2 reduces alkaloid accumulation in roots (Sui et al., 2018). Growing evidence indicates that genes related to the biosynthesis of active components also show tissue-specific expression patterns, PtMYC2 (PtbHLH73/112) is highly expressed in P. ternata tubers which are rich in components such as alkaloids and, nucleosides. Based on this, it is speculated that PtMYC2 (PtbHLH73/112) in P. ternata may respond to JA signals and regulate alkaloid synthesis in P. ternata. In the ABA treatment group, the expression levels of genes such as PtbHLH52/66/68/126 changed significantly, among which PtbHLH52 is a CUL4 homologous gene. In A. thaliana, DWA1 and DWA2 are two DWD protein components of CUL4-based E3 ligases, and they jointly act as negative regulators in ABA signal transduction (Lee et al., 2010); similarly, PtbHLH52 in P. ternata is also negatively regulated by ABA signals. In the SA treatment group, the expression of PtbHLH genes showed no obvious changes. This may be due to the insensitivity of P. ternata leaves to SA induction, resulting in insignificant changes at 1 h; significant differences may occur at later time points. Notably, the statistical power of the hormone-treated RNA-seq experiment was 0.48, which is below the commonly accepted threshold of 0.8. This indicates that the current number of biological replicates limits the ability to reliably detect genes with moderate expression changes. Therefore, we interpreted all non-significant expression results with caution. Increasing the number of biological replicates will be adopted in future related studies to improve the statistical power and detection capability.

High temperature, drought stress, and hormone induction have significant impacts on the growth of P. ternata. To further explore the role of PtbHLHs in the above environments, this study analyzed the expression profiles of 11 PtbHLHs under high temperature, drought stress, and hormone induction. Under high-temperature stress: Silencing LibHLH87 in Lilium reduced its basal heat tolerance, while LibHLH871 could activate LlEIN3 and LlHSFA2 to improve heat tolerance in Lilium (Wu et al., 2024). In P. ternata, PtbHLH9 showed a characteristic of significantly increased expression in response to high temperature, indicating that it may act as a core node in the heat tolerance regulatory network of P. ternata by activating the transcription of downstream heat shock proteins (HSPs) or genes related to stress-resistant metabolic pathways; in contrast, some genes (PtbHLH67/127) showed delayed responses, which may be associated with cell repair under long-term high-temperature stress. Under drought stress: Most genes exhibited an expression pattern of “decrease first, then increase, then decrease again”, which was basically consistent with the activation sequence of the plant osmotic adjustment mechanism. However, PtbHLH76 showed a relatively gentle response pattern, suggesting that it may have functional redundancy or a specific regulatory role in drought stress. At the hormone response level: The expression trends of PtbHLH genes after 1 h of MeJA, ABA, and SA treatments were basically consistent with the transcriptome data. Notably, PtbHLH48 was an exception—its expression showed no significant changes under the three hormone inductions in the transcriptome results, but was significantly highly expressed in qRT-PCR experiments. This inconsistency is mainly caused by independent biological samples and different detection sensitivity of the two techniques. As a homologous gene of HEC2, HEC1 has been found in A. thaliana to act as a local regulator of auxin and cell division protein responses to control female cell development in Arabidopsis (Schuster, Gaillochet & Lohmann, 2015); however, its association with other hormones has not been studied yet, which is worthy of in-depth exploration in subsequent research.

In summary, we systematically characterized the bHLH gene family specific to the medicinal plant P. ternata via multi-dimensional bioinformatic analysis and expression profiling, and screened key candidate genes responding to high temperature, drought, MeJA, ABA and SA. The unique evolutionary features, cis-element distribution and tissue/stress expression patterns of PtbHLHs identified here fill the research gap of bHLH transcription factors in this medicinal species. These results lay a foundation for uncovering the species-specific molecular regulatory mechanisms of PtbHLHs in abiotic stress tolerance and hormone signal transduction, and provide potential genetic targets for stress resistance improvement and molecular breeding of P. ternata. For future research, molecular biology techniques such as gene overexpression and gene knockout will be used to verify the potential biological functions of expression-supported candidate genes including PtbHLH9, PtbHLH85, PtbHLH73, PtbHLH7 and PtbHLH48 in P. ternata, as no direct functional validation was performed in the current study. Further experiments covering cold and salt stress treatments will also be supplemented to explore their expression response patterns. In addition, metabolite quantification of alkaloids and nucleosides under different treatments will be conducted in follow-up work to establish the correlation between the expression of PtbHLHs and the accumulation of medicinal active ingredients, which is a limitation of the present study. Another limitation is that only 18S rRNA served as the qRT-PCR reference gene, and no no-RT controls were included. Further assays with multiple reference genes and no-RT controls will validate these expression results.

Conclusions

The bHLH family has been widely characterized across plants. Here, 140 PtbHLH genes were identified from the P. ternata genome and divided into 24 subfamilies via phylogenetic analysis with A. thaliana. We systematically analyzed their chromosome distribution, gene structure, conserved domains, promoter cis-elements, collinearity, duplication events, GO/KEGG enrichment and protein-protein interactions. Tissue-specific expression patterns suggest diverse developmental functions of PtbHLH members in P. ternata. qRT-PCR under heat and drought stresses identifies PtbHLH9 as a heat-responsive gene and PtbHLH85 as a drought-responsive gene. Hormone treatment assays further identify key responsive genes: PtbHLH73 for MeJA, PtbHLH7 for ABA, and PtbHLH48 for MeJA, ABA and SA. This work characterizes the structural features and expression patterns of the PtbHLH family, provides clues for studying its regulatory mechanisms in abiotic stress and hormone responses, and offers insights for molecular breeding of high-quality P. ternata.

Funding Statement

This study was supported by the National Natural Science Foundation of China (Grant No. 81703652), the Natural Science Basic Research Program of Shaanxi Provincial Department of Science and Technology (Grant No. 2025JC-YBMS-1042), the Qinchuangyuan Traditional Chinese Medicine Industry Innovation Cluster & Joint Fund of Shaanxi University of Chinese Medicine (Grant No. L2024-QCY-ZYYJJQ-X194), and the Qinchuangyuan Traditional Chinese Medicine Industry Innovation Cluster Industry-University Cooperation Horizontal Project (Grant No. 303-206240051). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.

Additional Information and Declarations

Competing Interests

The authors declare that they have no competing interests.

Author Contributions

Xiaorui Wang conceived and designed the experiments, performed the experiments, analyzed the data, prepared figures and/or tables, authored or reviewed drafts of the article, and approved the final draft.

Huirong Li conceived and designed the experiments, analyzed the data, authored or reviewed drafts of the article, and approved the final draft.

Zimo Li performed the experiments, analyzed the data, prepared figures and/or tables, and approved the final draft.

Han He performed the experiments, prepared figures and/or tables, and approved the final draft.

Ziyu Xia performed the experiments, analyzed the data, prepared figures and/or tables, and approved the final draft.

Yajun Liu performed the experiments, analyzed the data, prepared figures and/or tables, and approved the final draft.

Xinghang Cai conceived and designed the experiments, authored or reviewed drafts of the article, and approved the final draft.

Wenjing Huang conceived and designed the experiments, authored or reviewed drafts of the article, and approved the final draft.

Xiaochun Sun conceived and designed the experiments, authored or reviewed drafts of the article, and approved the final draft.

Data Availability

The following information was supplied regarding data availability:

The raw data are available in the Supplemental Files.

The data is available at Figshare:

- wang, xiaorui (2026). Transcriptome data of Pinellia ternata under MeJA, ABA and SA treatments. figshare. Dataset. https://doi.org/10.6084/m9.figshare.31697926.v1.

The raw sequence data are available at NGDC BioProject: PRJCA016404.