Genome-Wide Identification and Characterization of bHLH Transcription Factors and Their Expression Profile in Potato (Solanum tuberosum L.)

Abstract

The bHLH gene family, one of the most abundant transcription factor families in plants, plays crucial roles in stress resistance, growth, and development. To explore the characteristics of the potato bHLH gene family members, this study identified and analyzed a total of 134 bHLH genes. Using bioinformatics approaches, we examined their physicochemical properties, conserved structural domains, motifs, and cis-acting elements. Additionally, a phylogenetic analysis was conducted, comparing the bHLH proteins of potato with those of the model plant Arabidopsis thaliana. The study also investigated the expression patterns of StbHLH genes under different environmental conditions and growth stages. The potato bHLH gene family is enriched with various cis-acting elements associated with stress response and plant hormone signaling. The expression patterns of StbHLH genes varied significantly across different conditions, revealing their potential roles in stress resistance and developmental processes. For example, under drought and re-watering treatments, distinct expression patterns were observed, with specific genes showing upregulation or downregulation at different time points. StbHLH025 regulates tissue development and stress response in potato. These findings not only reveal the diversity and complexity of the potato bHLH gene family but also provide valuable insights for future research into the functions of StbHLH genes, particularly their roles in potato stress resistance and developmental processes.

1. Introduction

As the fourth most important staple crop globally, potato (Solanum tuberosum L.) plays a pivotal role in ensuring food security and sustaining agricultural economies. Its versatility extends beyond human consumption to livestock feed and industrial applications, driven by its high yield potential and adaptability to diverse agroecological conditions [1]. However, potato cultivation is increasingly threatened by abiotic stresses, particularly drought and soil salinity, which severely impair growth and productivity [2]. Drought stress alone can reduce tuber yields by over 30%, causing catastrophic impacts in arid and semi-arid regions where water scarcity exacerbates agricultural vulnerabilities [3]. For instance, recurrent drought events in major potato-growing areas have led to substantial economic losses, and have destabilized local food systems [4,5]. Addressing these challenges requires not only advanced breeding strategies but also a deeper understanding of molecular regulators, such as transcription factors, that orchestrate stress adaptation mechanisms critical for crop resilience.

Within these regulators, the basic helix-loop-helix (bHLH) transcription factors are considered the second-largest family of transcriptional regulators in plants. Characterized by a conserved bHLH domain comprising ~60 amino acids, this protein family includes two functional regions: (1) a basic region (13–17 residues) responsible for DNA binding and (2) a helix-loop-helix (HLH) region (~40 residues) mediating protein dimerization [6,7]. Functionally, bHLH proteins regulate diverse processes, including growth, development, and stress responses, positioning them as promising targets for engineering stress-tolerant crops [8]. Genome-wide analyses reveal species-specific variation in bHLH family size, with Arabidopsis thaliana (162 genes), Oryza sativa (167), and Zea mays (208) exhibiting distinct gene numbers [9,10,11]. Phylogenetically, plant bHLH proteins are classified into six major groups (A–F), which are further subdivided into 15–26 subgroups based on evolutionary relationships and DNA-binding specificities. Notably, this classification system remains less resolved compared to the well-defined animal bHLH framework, reflecting the complexity of plant-specific functional diversification shaped by adaptive evolution to diverse environments [12]. While studies in model species like Arabidopsis and rice have elucidated roles of bHLH genes in photomorphogenesis (e.g., PIFs), iron homeostasis (e.g., FIT), and drought tolerance (e.g., AtbHLH68), translating these insights to crops like potato requires species-specific investigations [13].

Previous studies have identified 124 StbHLH genes in potato, which are randomly distributed across its 12 chromosomes. Gene Ontology (GO) annotations revealed that StbHLH proteins participate in diverse biological processes, including biosynthetic processes, metabolism, stress response, and tissue/organ development [14]. StbHLH genes exhibit broad expression across multiple tissues and are dynamically regulated by abiotic stresses, including salinity, drought, and elevated temperatures. Beyond stress responses, these transcription factors integrate light, cold, and hormonal signals modulating critical biological processes such as anthocyanin biosynthesis, epidermal cell fate determination, and root/floral developmental patterning. Mechanistically, StbHLH proteins bind to specific cis-elements, DNA sequences in the promoters of downstream genes, to orchestrate transcriptional networks that enhance resilience. For instance, StbHLH47 has been identified as a negative regulator of drought tolerance in potato, highlighting the functional diversity within this family [15]. Furthermore, StbHLH-mediated regulation of root architecture, particularly through modulating root elongation and branching patterns, enhances water foraging capacity under drought conditions. Despite these advances, most studies have focused on individual StbHLH genes, leaving systemic family-level analyses and their translational potential underexplored.

To address this gap, we identified 134 putative StbHLH genes in the potato genome and performed a detailed analysis of their phylogenetic relationships, gene structures, conserved motifs, and chromosomal distributions. Our study further revealed tissue-specific expression patterns and dynamic transcriptional responses under abiotic stresses, highlighting their potential roles in drought adaptation and recovery processes. For example, distinct StbHLH clades exhibited preferential expression in roots or leaves during drought-rehydration cycles, suggesting specialized functional modules. Unlike prior studies in Arabidopsis and rice that focused on isolated roles in stomatal regulation or osmotic adjustment, this systemic approach uncovers both conserved and lineage-specific regulatory hubs. Future efforts will prioritize functional validation using CRISPR-based editing to dissect mechanisms underlying drought tolerance, such as the interplay between StbHLH genes and ABA signaling pathways. By bridging molecular insights with agricultural needs, this work provides the knowledge necessary for selecting candidate genes, which can be used in potato improvement experiments to develop cultivars.

2. Materials and Methods

2.1. Identification and Characterization of bHLHs

The chromosome location information for each StbHLH gene was obtained from the potato genome database (http://spuddb.uga.edu/index.shtml, accessed on 18 June 2023). The software MapChart (version 2.32) was employed to graphically represent the genes on the chromosomes [16]. The complete genomic sequences, predicted amino acid sequences, and transcript sequences for all potato genes were obtained from the same potato genome database. Conserved domains within the potato protein-coding genes were detected using HMMER and the PFAM databases (http://pfam.sanger.ac.uk, accessed on 2 September 2023). The putative StbHLHs in S.tuberosum were identified through protein-protein BLAST (version 2.16.0) using the 109 Arabidopsis bHLH (AtbHLH) amino acid sequences, with an E-value cutoff of 10⁻¹⁰.

For the construction of a phylogenetic tree, multiple sequence alignments of the bHLH domains of the StbHLHs were performed using MEGA 5.0, with 1000 bootstrap replicates to ensure statistical robustness [17]. The molecular weight (MW) and isoelectric point (PI) of each bHLH protein were calculated using the online ExPASy Bioinformatics Resource Portal (https://web.expasy.org/compute_pi/, accessed on 16 October 2024) with default parameters. The subcellular localization was predicted by uploading protein sequences to the Plant-mPLoc online tool (http://www.csbio.sjtu.edu.cn/bioinf/plant-multi/, accessed on 16 October 2024).

The MCScanX toolkit was used to compare the sequences from the potato genome and analyze tandem repeats and segmental repeats [18]. Additionally, KaKs_Calculator 2.0 was used to calculate the ratio of nonsynonymous replacement rate (Ka) to synonymous replacement rate (Ks) for protein-coding genes [19], serving as an indicator of nucleic acid molecular evolution. A Ka/Ks ratio less than 1 suggests negative selection, a ratio greater than 1 implies positive selection, and a ratio equal to 1 indicates neutral selection.

2.2. Analysis of Conserved Motif Distribution and Gene Structure

The MEME suite (version 5.4.1) (https://meme-suite.org/meme/tools/meme, accessed on 20 October 2024) was utilized to analyze conserved motifs for each StbHLH. The parameters for motif identification were configured as follows: the motif discovery mode was set to classic mode; the site distribution was chosen as zero or one occurrence per sequence (zoops); and the number of motifs to identify was set to 10. The number of amino acids and length of coding sequences (CDS) were calculated using the TBtools (version 2.154) software. To further visualize the motif composition and gene structure, integration analysis was conducted using TBtools (version 2.154) [20]. The online tool PlantCARE (http://bioinformatics.psb.ugent.be/webtools/plantcare/html/, accessed on 20 October 2024) was used to analyze the cis-acting elements in the promoter sequences of StbHLH genes, examining the genomic region 2 kb upstream of the ATG start codon.

2.3. Gene Expression Profile Analysis

The expression of each StbHLH gene in different varieties and different tissues was screened from potato genome database (http://spuddb.uga.edu/index.shtml, accessed on 25 October 2024), and heat map analysis and cluster analysis were carried out by using Heatmap package in R software (version 4.0.5). The data were individually mapped to the potato genome using HISAT2 (version 2.0.5) with default parameters [21]. Transcript abundance and differential gene expression were assessed using the featureCounts program and the transcripts per kilobase million method. The DESeq2 R package (version 1.16.1) was applied to identify differentially expressed genes, defined as those with a p < 0.05 and an absolute log2 (fold change) value > 1.

2.4. Weighted Gene Co-Expression Network Analysis (WGCNA)

The R package WGCNA is to construct gene co-expression network based on gene expression data. Here, based on the correlation intensity score of transcription factors to target genes, this method establish a gene-gene pair interaction network. Log2 normalized fragments per kilobase of exon model per million mapped fragments (FPKM) values were used to construct co-expression networks with the WGCNA package in R [22]. An adjacency matrix was constructed using a soft threshold power of 4. Network interconnectedness was measured by calculating the topological overlap using the topological overlap measure (TOM) dist function with a signed TOM. Average hierarchical clustering using the hclust function was performed to group the genes based on the topological overlap dissimilarity measure (1-TOM) of their connection strengths. Network modules were identified using a dynamic tree cut algorithm with minimum cluster size of 15 and merging threshold function at 0.25. To visualize the expression profiles of the modules, the eigengene (first principal component) for each module was plotted using ggplot2 in R. To identify hub genes within the modules, the module membership (MM) for each gene was calculated based on the Pearson correlation between the expression level and the module eigengene. Genes within the module with the highest MM are highly connected within that module. To relate the physiology measurements with the network, the module eigengenes were correlated with the physiology data. Correlations were performed for each physiology trait separately using the mean values at each time point to associate the diel patterns between the physiology and eigengenes. Gene connectivity within a module represents the regulatory relationship between a gene and other genes. A higher connectivity reflects a greater regulatory role of the gene in the module, and the more likely it is to be a potential hub gene. We screened the top 10 genes in the connectivity of each module and then selected genes with a cut-off value > 0.25 of the weight parameter weight and gene connection numbers > 5 as candidate genes for the construction of a gene interaction network. Hub genes were identified based on their eigengene connectivity. Networks were visualized using Cytoscape 3.8.2 software (https://cytoscape.org, accessed on 25 October 2024).

2.5. Isolation of RNA and Quantitative Real-Time PCR Analysis

This study used tissue-cultured seedlings of the tetraploid cultivated potato (Solanum tuberosum L. cv. Atlantic) as materials to systematically conduct abiotic stress treatments and dynamic sampling during the growth and development stages. In the stress experiment module, plants with uniform growth status were subjected to three treatments: (1) 100 mM NaCl solution to simulate salt stress, (2) 200 mM mannitol solution to simulate osmotic stress, and (3) 10% (w/v) PEG6000 solution to simulate drought stress. Each treatment group was set with four time gradients of 1, 2, 4, and 6 h, and included three biological replicates. Concurrently, throughout the entire growth cycle, leaves, petioles, stems, stolons, roots, and tubers of the potato were regularly collected and sampled. All samples were flash-frozen in liquid nitrogen and stored at −80 °C for subsequent analysis.

Total RNA was extracted following the instructions of the RNAprep Pure Plant Total RNA Extraction Kit (DP441) from Tiangen Biotech (Beijing, China) Co., Ltd. cDNA (Complementary DNA) that was reverse transcribed using the FastKing RT Kit (With gDNase) was used as template, and SYBR Green SuperMix Plus (TIANGEN BIOTECH CO., LTD, Beijing, China) served as the fluorescent dye. Gene-specific primers for qPCR were designed using Primer5 according to the corresponding sequence. Primers were synthesized by Sangon Biotech (Shanghai, China) Co., Ltd. The volume of the reaction system was 20.0 μL, including 10.0 μL of 2× TIANGEN^® SYBR qPCR SuperMix Plus, 0.6 μL of each primer, 6.8 μL of RNase-free water, and 2.0 μL of template. The PCR cycle was: 10 min at 95 °C, 40 cycles of 15 s at 95 °C, and 32 s at 60 °C. A Light Cycler 480 instrument (Bio-Rad, Hercules, CA, USA) was used for qRT-PCR. The potato internal reference genes elongation factor 1α (StEF1α) was used to normalize the gene expression level using the geometric mean, and the primer sequence was obtained from previous studies. Each gene was analyzed with three technical replicates. The relative expression level (2^−ΔΔct) of untreated control plants was set to 1.

2.6. Statistical Analyses

At least three independent biological replicates were used for each analysis. Statistical analysis was performed using the SPSS 22.0 software (SPSS Inc., Chicago, IL, USA). Differences in means among treatments were calculated using Duncan’s multiple range test. ggplot2 was used to generate plots. Differences with p < 0.05 were considered statistically significant.

3. Results

3.1. Identification and Characterization of StbHLH Genes

To identify bHLH gene family members in the potato genome, sequence alignments were performed between the bHLH genes of Arabidopsis thaliana and those of potato using two BLASTP searches. BLASTP is a powerful tool for identifying regions of local similarity between sequences. After removing redundant sequences, a total of 134 bHLH genes were confidently identified and selected from the potato genome. These genes were sequentially named from StbHLH001 to StbHLH134 based on their chromosomal positions in the assembled potato genome (Table S1). An in-depth analysis was conducted to summarize the fundamental characteristics of these genes, including molecular weight (MW), isoelectric point (pI), coding sequence (CDS) length, and predicted subcellular localization. The length of the deduced StbHLH proteins ranged from 86 amino acids (aa) in StbHLH067 to 929 aa in StbHLH127, with an average protein length of 375.47 aa. The calculated isoelectric points and molecular weights for the StbHLH proteins ranged from 4.55 (StbHLH058) to 11.52 (StbHLH003) for pI, and from 9.80 kDa (StbHLH067) to 101.69 kDa (StbHLH127) for MW (Table S1). Regarding subcellular localization, specific predictions were made for StbHLH124 and StbHLH066, suggesting that they might be located in the endoplasmic reticulum (Table S1). Additionally, StbHLH104 was predicted to localize to both the cell membrane and nucleus. For the remaining StbHLH proteins, the nucleus was predicted as their primary localization site. These predictions provide valuable insights into the potential functional roles of these proteins within the cell.

3.2. Chromosome Distribution and Gene Replication of StbHLH Family Genes

The distribution of StbHLH genes (StbHLH001 to StbHLH134) across the potato chromosomes reflects the evolutionary diversification and complexity of the bHLH gene family. The number of StbHLH genes per chromosome varies significantly, ranging from 2 on Chromosome 11 to 19 on Chromosome 01, with an average of 11.17 genes per chromosome. This variation highlights the intricate nature of the gene family. A clustering tendency is evident among these genes, as seen in several groups located on specific chromosomes. For example, Chromosome 01 contains StbHLH004 to StbHLH019, Chromosome 02 has StbHLH020 to StbHLH033, Chromosome 06 includes StbHLH078 to StbHLH086, and Chromosome 09 harbors StbHLH108 to StbHLH116 (Figure 1A). This clustering pattern suggests that these genes may have undergone segmental duplication events leading to gene family expansion. Chromosome 01 contains the highest number of StbHLH genes, with 19 genes accounting for 14.18% of the total identified StbHLH genes. Chromosome 04 follows with 16 genes (11.94%), while Chromosome 11 has the fewest, with only 2 genes (1.49%) (Figure 1B).

A total of 78 StbHLH genes exhibiting tandem duplications were identified within the potato genome, and these were categorized into 28 distinct clusters (Figure 1A). These findings suggest that tandem duplication has played a key role in expanding the StbHLH gene family. Our analysis revealed a broad range of Ka/Ks, spanning from 0.10 to 2.63 across 303 gene pairs. Among these, 122 gene pairs exhibited a Ka/Ks greater than 1, indicating the influence of positive selection on these genes. Conversely, 181 gene pairs had a Ka/Ks less than 1, suggesting negative selection. This distribution indicates that the majority of gene pairs in our sample are under negative selection, highlighting the dominant role of purifying selection in shaping these genetic sequences (Table S2).

3.3. Phylogenetic Analysis of StbHLH Genes

The phylogenetic analysis of the StbHLH gene family in potato, combined with bHLH genes from Arabidopsis thaliana, provides valuable insights into the evolutionary relationships and potential functions of these transcription factors. By examining the full-length protein sequences of 134 StbHLH genes from potato and 125 bHLH genes from A. thaliana, we constructed a comprehensive phylogenetic tree that classified the StbHLH genes into eight distinct subgroups. Group I is the largest subgroup, containing 46 genes, while Group V is the second largest with 37 genes. Group IV, II, and VI are moderately sized, with 17, 14, and 11 genes, respectively. Group VII, III, and VIII are smaller, each with fewer than 10 genes. Notably, Group VIII contains only two genes, StbHLH107 and StbHLH076, which exhibit relatively low similarity to genes in other subgroups (Figure 2). This suggests that these two genes may have significantly diverged from the rest of the family or could represent a more ancient or distinct lineage within the bHLH family. Their placement in a single clade within Group VIII implies a close evolutionary relationship between them, potentially sharing unique functions or regulatory roles in the potato plant.

3.4. Gene Structure, Motif Composition and Protein Interaction Analysis of StbHLH Genes

Motifs are sequences of amino acids that have evolved to perform specific biological functions and are often associated with particular structural or functional domains. To explore the functional regions within the StbHLH proteins, we utilized the MEME online tool to analyze the amino acid motifs in the 134 StbHLH proteins (Figure 3). We examined the distribution of these conserved motifs in the context of their phylogenetic relationships (Figure 3). Our analysis identified 10 distinct conserved motifs (

Table 1. Conserved motifs of bHLH proteins in potato plant.

Motif	Length (aa)	Amino Acid Conserved Sequence
Motif 1	27	HSLAERRRREKJNERMKALQELVPNCN
Motif 2	21	KTDKASMLDEAINYIKELQEQ
Motif 3	15	DYIHVRARRGQATDS
Motif 4	21	RLMEALESLGLDVLHANISTV
Motif 5	29	RSSHIAVERNRRKKMNELFSELRSLLPPS
Motif 6	20	VEFLSMKLATVNPRLDFNJD
Motif 7	29	VCIPTPSGVVELGSTEVIPENLELVQQVK
Motif 8	26	PEIEVKIIGDDAMIRVQSEKKPGPLL
Motif 9	27	EEAMQDLRSRGLCLVPISCTTAISSST
Motif 10	42	NVTDTEWFYLMSMAQSFPVGEGLPGKAFSSGSHIWLTGAQEL

). We found that most StbHLH genes clustered in the same phylogenetic clade shared similar motif compositions at comparable positions. This pattern suggests a strong correlation between motif composition and biological function, indicating that proteins within the same clade likely perform analogous roles in the biological system. Notably, Motifs 1 and 2 were the most prevalent and were identified in the majority of the StbHLH proteins. Most StbHLH proteins contained at least 3–4 conserved motifs, although a few specific proteins deviated from this trend. These findings provide significant insights into the functional conservation and divergence of the StbHLH protein family.

Structural variations within genes are fundamental to the evolution of gene families and are crucial for understanding the genetic diversity and environmental adaptability of plants. To explore this diversity in gene structure, we used TBtools (version 2.154) to characterize the architecture of StbHLH genes, including untranslated regions (UTRs), exons, and introns. We found that genes clustered within the same category tended to exhibit similar structural patterns. Our analysis revealed a wide range in the number of exons and introns among the StbHLH genes. StbHLH124 contained the most exons and introns among the genes studied. This was followed by StbHLH041 and StbHLH082, each containing 11 exons and 11 introns. Interestingly, several StbHLH genes, such as StbHLH004, StbHLH005, StbHLH010, StbHLH015, StbHLH035, StbHLH051, StbHLH056, StbHLH093, StbHLH058, StbHLH099, StbHLH101, StbHLH105, and StbHLH124, lacked UTRs despite having exons and introns. This absence suggests a distinct evolutionary trajectory for these genes, as shown in Figure 3. While there were some variations in the composition among different StbHLH classes, the overall genetic structure within each class was relatively conserved. The specific structural features of these classes could be attributed to the functional divergence observed among them. These structural differences may underlie the varied physiological roles that these genes play within the plant, potentially influencing their regulatory mechanisms, expression patterns, and interactions with other cellular components.

3.5. Analysis of the Promoter Region Cis-Regulatory Element

The 2000-bp upstream sequences of the StbHLH genes were extracted to predict cis-acting elements. The promoter region of genes plays a crucial role in regulating gene expression, containing various cis-regulatory elements that respond to different environmental and hormonal signals. In the StbHLH gene family of potato plants, an in-depth analysis of these promoter regions revealed three main types of cis-regulatory elements: hormone response elements, stress response elements, and MYB binding sites. We analyzed these three types of cis-regulatory elements within the promoter regions. Hormone response elements included those for abscisic acid (ABA), auxin (Aux), gibberellin (GAs), methyl jasmonate (MeJA), and salicylic acid (SA). Stress response elements included those induced by wounding, light, low temperatures, and general stress defense mechanisms. Additionally, we examined MYB binding sites, which are crucial for processes like flavonoid biosynthesis, light response, and the specific binding associated with MYBHv1. Our findings indicate that among the StbHLH genes, 18 possess elements responsive to light, while 48 are equipped to handle low temperatures. Furthermore, 50 genes demonstrate involvement in defense mechanisms and stress responses. Notably, each StbHLH gene can be induced by one to five different hormones. Moreover, 84 members have MYB binding sites, with some featuring up to three distinct binding sites (Table S3). This comprehensive analysis of the promoter’s cis-regulatory elements suggests that the bHLH gene family in potato plants has a sophisticated regulatory capacity. It can respond to a variety of hormonal signals and abiotic stresses, with MYB likely playing a role in the transcriptional regulation of StbHLH genes. These elements are poised to dictate the transcriptional activity and overall stress responsiveness of the bHLH gene family when faced with adverse conditions.

3.6. Expression Patterns of bHLH Genes in Potato Plant Under Different Tissues

The differential expression profiling of StbHLH genes across various tissues of the potato plant was conducted through a meticulous screening and analysis process. To validate gene association patterns and identify highly coordinated genes among different samples, a comprehensive matrix consisting of 34 samples (Table S4) and 134 genes (Table S5) was extracted from the database. This matrix served as the foundation for co-expression network analysis using the WGCNA method. The input for the co-expression network construction was a standardized sample gene expression profile matrix. By constructing a hierarchical clustering tree with 133 genes across 34 tissue samples from the potato, it was observed that there were no conspicuous outliers (Figure S1). Notably, StbHLH014 was not expressed in any tissue. To enhance the network for scale-free characteristics, a β value of 4 was chosen for constructing the co-expression network. By the dynamic tree cutting method for module identification, a total of five distinct modules were identified (Figure S2). Genes that did not fit into any specific module were categorized under a gray module and were subsequently excluded from further analysis. The TOM heatmap was crafted to reflect the interactions among the five identified modules (Figure S3). Callus tissues (10 and 11 week old) were generated from leaves and stems. The blue module, comprising 23 genes, demonstrated an association with callus tissues (SRR122123 and SRR122113). The brown module, representing 20 genes, showed a significant correlation with root tissue (ERR029917). Within this module, StbHLH089 exhibited a strong relationship with StbHLH028 and StbHLH026. The green module, with 17 genes, was linked to dormant tuber tissue (SRR1039535). The turquoise module, encompassing 27 genes, was highly associated with various flower parts, including sepals (SRR122121), whole mature flowers (SRR124131), petals (SRR124132), stamens (SRR122111), and carpels (SRR122137). The harvested tubers were stored under standard commercial conditions until sprouting was present (non-dormant). The yellow module, consisting of 18 genes, was found to be highly associated with the non-dormant meristem (SRR1584267). Notably, StbHLH089, StbHLH058, and StbHLH096 exhibit high expression levels across all tissues examined (Figure 4). These findings underscore the potential roles of these genes in diverse physiological processes and highlight their potential as biomarkers for tissue-specific functions or responses in the potato plant.

3.7. Expression Patterns of StbHLH Genes in Potato Plant Under Abiotic Stress

To investigate the role of StbHLH genes in the potato plant’s response to abiotic stress, we analyzed RNA-seq data to assess the expression levels of the 134 bHLH genes in potato leaves under water stress, salt, mannitol, and heat treatments. The expression patterns of these genes varied significantly across different stress conditions. Compared to the control group, most StbHLH genes exhibited reduced expression under water stress. Conversely, under salt and mannitol stress, the majority of these genes showed increased expression levels relative to the control. Cluster analysis revealed that the 134 StbHLH genes could be broadly categorized into three groups: genes responsive to water stress (25 genes), those responsive to salt and mannitol stress (67 genes), and those responsive to heat stress (42 genes). This classification provides a clearer understanding of the specific stress responses of the StbHLH gene family in potato plants (Figure 5).

3.8. Expression Profiles of StbHLHs Under Drought and Re-Watering Treatment

To identify candidate genes differentially expressed under drought and re-watering treatments, this study utilized correlation heatmaps for screening and analysis. The 134 potato bHLH genes exhibited distinct expression patterns in response to these treatments (Figure 6). Specifically, under leaf drought and re-watering at two time points, there were 14 and 1 upregulated genes, respectively, and 19 and 8 downregulated genes, respectively. Similarly, under tuber drought and re-watering at two time points, there were 4 and 1 upregulated genes, respectively, and 9 and 14 downregulated genes, respectively. In the leaves, the expression levels of StbHLH045, StbHLH120, StbHLH079, StbHLH074, StbHLH010, and StbHLH038 were consistently lower than those of the control group throughout the drought and re-watering process. In the tubers, StbHLH024 showed higher expression than the control group under drought, which then decreased after re-watering, indicating a positive response to drought. Conversely, the genes StbHLH038, StbHLH105, and StbHLH125 were downregulated under drought but showed increased expression after re-watering, suggesting a negative response to drought (Figure 6).

3.9. StbHLH025 Regulates Tissue Development and Stress Response in Potato

To systematically elucidate the dual functions of the StbHLH025 in potato growth, development, and stress response, this study employed a multi-dimensional analysis integrating transcriptomic data and qRT-PCR technology (

Table 2. Primer sequences used for StbHLH025 qRT-PCR.

Gene Name	Forward Primer Sequence (5′-3′)	Reverse Primer Sequence (5′-3′)
StEfla (reference gene)	CAAGGATGACCCAGCCAAG	TTCCTTACCTGAACGCCTGT
StbHLH025	AATGCTCAATGGCCCTCCAA	CCCTGCGCATTTTCTCCCTA

). Preliminary screening based on the transcriptomes of diploid materials ‘DM1-3516R44’ and ‘RH89-039-16’ revealed high expression levels of this gene in flowers and leaves (Figure 7A). Further validation using the tetraploid cultivar ‘Atlantic’ for tissue-specific expression showed significant organ-specific expression patterns: the highest expression in leaves (6.41 ± 1.14), followed by stolons (3.60 ± 1.43), and the lowest in tubers (0.01 ± 0.002), which corroborates the findings from the initial data across genotypes (Figure 7B). The StbHLH025 is induced by various biotic and abiotic stresses (Figure 7C). In response to abiotic stresses, the gene exhibits dynamic regulation: under PEG6000 treatment, it peaked at 234.19-fold at 1 h and then declined; under NaCl stress, its expression surged to 16.92-fold at 1 h and sharply dropped to 3.02-fold at 2 h; under mannitol treatment, it triggered biphasic fluctuations, increasing to 3.56-fold at 0–1 h, decreasing to 1.54-fold at 2 h, and then rising again to 3.08-fold at 4 h (Figure 7D).

4. Discussion

In the research domain of potato bHLH transcription factor family, prior studies have established critical foundations through genome-wide identification, meticulous gene structure characterization, in-depth phylogenetic analysis, and comprehensive expression profiling, providing invaluable preliminary data for elucidating the functions of this pivotal gene family [14]. However, the current study achieves substantial advancements and expansions. We not only expanded the potato bHLH repertoire by identifying a larger cohort of family members but also uncovered coordinated regulatory mechanisms of specific bHLH members in drought, salt, and heat stress responses. Furthermore, WGCNA revealed tissue-specific functional divergence among StbHLH genes. In this research, we employed bioinformatics tools to conduct an in-depth, genome-wide identification of the bHLH gene family within the potato genome, uncovering a total of 134 bHLH gene family members. Comparative analysis with other reported plant species revealed quantitative differences in the size of the bHLH gene family among them: the potato has a relatively lower number of bHLH genes compared to Arabidopsis, rice, and maize, with 28, 33, and 74 fewer members, respectively [9,11,23]; whereas in comparison to melon, sweet potato, pepper, and sugarcane, the potato exhibits a higher number of bHLH gene members, with an additional 16, 24, 27, and 97 members, respectively. These comparative data provide significant clues regarding the potential uniqueness and diversity of the potato bHLH gene family in terms of evolution and function [24,25,26,27]. This insight may help elucidate the distinctive roles these genes could play in the biology and adaptation of the potato species. This uneven distribution could have implications for the functional diversification of the StbHLH genes, with gene-rich regions potentially associated with specific biological pathways or responses to environmental stimuli. The clustering of these genes might also provide opportunities for co-regulation or functional redundancy within the potato genome. Understanding the distribution and evolutionary patterns of the StbHLH family can offer valuable insights into the genetic architecture and adaptive strategies of potato plants.

This study identified 134 StbHLH genes in potato distributed across 12 chromosomes, with 25 genes responsive to water stress, 67 to salt stress, and 42 to heat stress. In sweet potato (Ipomoea batatas), 227 IbbHLH genes were localized to 15 chromosomes, including 17 segmental duplicate gene pairs and 5 pairs of tandemly duplicated genes within the IbbHLH family, with 12 IbbHLH genes involved in salt (150 mM NaCl) and drought (20% PEG6000) stress regulation, and 20 IbbHLH genes were regulated by cold stress [25,28]. In Carthamus tinctorius, 120 CtbHLH genes were identified and distributed across all 12 chromosomes, among which 15 genes exhibited significant expression profile changes over a 24-h period under salt stress (NaCl), drought stress (PEG6000), and hormone treatments (ABA and MeJA) [29]. For melon (Cucumis melo), 118 CmbHLH genes were broadly distributed across 12 chromosomes, with 38 genes showing duplication events [27]. These findings suggest that non-uniform chromosomal distribution and gene duplication events may enhance plant adaptability to complex environmental conditions through functional redundancy or sub functionalization.

Analysis of the physicochemical properties of the proteins encoded by the identified potato bHLH genes reveals a range of amino acid numbers from 86 to 929 aa, a molecular weight variation of 9.80 to 101.69 kDa, and a theoretical pI range of 4.55 to 11.52. Compared to the physicochemical properties of bHLH proteins in other species, the range of amino acid numbers and molecular weights in potato is broader than that found in foxtail millet (amino acids 79 to 897 aa, molecular weight 9.03 to 97.04 kDa, and pI 4.56 to 12.03) [30]. However, it is narrower than the ranges observed in sunflower (amino acids 77 to 731 aa, molecular weight 9.10 to 52.92 kDa) [31] and peanut (amino acids 90 to 1166 aa, molecular weight 9.97 to 133.36 kDa) [32]. This indicates that there is considerable variation in the distribution of bHLH gene family members on chromosomes and in their physicochemical properties among different species.

Analysis of the promoter regions of the potato bHLH gene family revealed a variety of cis-acting elements related to hormone response, light response, stress response, and transcription factor binding sites. The hormone response elements predominantly include those responsive to abscisic acid, gibberellins, and auxins. For example, under drought stress, FtbHLH3 enhances photosynthetic efficiency and upregulates the expression of key genes in the ABA signaling pathway, the proline biosynthetic pathway, the ROS scavenging system, and the drought-responsive pathway [33]. SlbHLH22 also improved tomato plant stress resistance by inducing the expression of genes involved in flavonoid biosynthesis [34]. ZmPIF1 is a positive regulator of root development, ABA synthesis, signaling pathways, and drought tolerance [35]. AtbHLH68 may positively respond to drought stress through ABA signaling and by regulating ABA homeostasis in Arabidopsis thaliana [36]. PebHLH35 in transgenic Arabidopsis enhances drought resistance by lowering stomatal density, opening, transpiration rate, and water loss, while boosting chlorophyll levels and photosynthesis rate [37]. OsbHLH148 also enhances drought tolerance by interacting in the jasmonate signaling pathway [38]. The released OsbHLH148 activates drought tolerance genes, including OsDREB1s [39]. This study on the identification and analysis of the potato bHLH gene family lays a foundational basis for further investigation into their functions and molecular mechanisms, which could be instrumental in developing strategies for crop improvement and stress tolerance.

Bioinformatic analysis of cis-regulatory elements in the promoter regions of potato bHLH genes revealed a diverse array of functional motifs associated with hormone responses (e.g., ABA, GA3, and IAA), light signaling, stress adaptation, and transcription factor binding. Notably, hormone-responsive elements were prominently enriched, reflecting potential roles in mediating phytohormone signaling and environmental adaptation. ABA-responsive elements, linked to drought, salinity, and cold stress responses, were widely distributed, consistent with studies showing drought- and ABA-inducible expression of PebHLH35 in Populus euphratica [37]. GA3- and IAA-related motifs, implicated in growth regulation and cell elongation, further aligned with functional evidence, such as AtbHLH68 in Arabidopsis thaliana, which modulates lateral root development and drought responses via ABA signaling [36]. The genome-wide identification and expression profiling of potato bHLH transcription factors, coupled with these promoter features, suggest that this gene family orchestrates crosstalk between hormonal pathways (e.g., ABA-GA3-IAA interplay) and stress adaptation mechanisms, providing a molecular framework for their roles in balancing growth and stress resilience.

In addition to hormone-responsive elements, the promoter regions of potato bHLH genes are enriched with light-responsive cis-regulatory elements, underscoring their potential roles in photoperception and photomorphogenesis. Light, a pivotal environmental cue, regulates diverse physiological processes such as photosynthesis, photoperiodic flowering, and shade avoidance. Mechanistically, bHLH transcription factors integrate light signals by interacting with photoreceptors. For instance, members of the bHLH VIIa subfamily, including Phytochrome-Interacting Factors (PIFs), act as negative regulators of photomorphogenesis by repressing light-triggered developmental transitions and maintaining etiolation in darkness [40]. Beyond signal transduction, bHLH factors directly modulate photosynthetic efficiency. Studies reveal their regulatory roles in light-harvesting complex (LHC) protein expression, critical for photosystem I assembly. Salt stress-induced suppression of LHC genes, mediated by bHLH activity, highlights their dual roles in stress adaptation and photosynthetic optimization [7]. Conversely, overexpression of SlPRE5, a bHLH gene in tomato, reduces chlorophyll content, impairing photosynthetic capacity [41]. Furthermore, bHLH-mediated photoperiodic regulation is exemplified by PIF4, which integrates temperature and light cues to control flowering timing [13]. These findings position potato bHLH genes as central hubs coordinating light signaling, photosynthetic homeostasis, and developmental plasticity under fluctuating environmental conditions.

The diversity of cis-regulatory elements in the promoter regions of potato bHLH genes collectively underscores their multifunctional roles in coordinating plant growth, hormone signaling, light responses, and stress adaptation. These elements likely orchestrate dynamic transcriptional regulation, enabling precise control of downstream processes such as energy synthesis (e.g., photosynthetic optimization via LHC regulation), biotic/abiotic defense mechanisms (e.g., ABA-mediated drought resilience), and developmental plasticity (e.g., photoperiodic flowering). By integrating hormonal cues (ABA, GA3, IAA) with environmental signals (light, stress), the StbHLH family forms a regulatory nexus that balances growth-defense trade-offs and adapts to fluctuating conditions. For instance, light-responsive PIF-like factors and stress-inducible ABA modules may synergize to optimize resource allocation under combined light and drought stresses. This multi-layered regulatory network highlights the evolutionary refinement of cis-regulatory landscapes in shaping plant fitness, providing a molecular blueprint for improving agronomic traits in potato through targeted manipulation of bHLH-mediated pathways.

5. Conclusions

In this study, we conducted a systematic, genome-wide analysis of the bHLH gene family in potatoes, identifying a total of 134 members across 8 distinct categories. Subsequently, we undertook a detailed examination of the StbHLH genes, encompassing their chromosomal localization patterns under various biological conditions, gene duplication events, gene structure, and motif composition, as well as the cis-regulatory elements within their promoter regions and their expression profiles. StbHLH transcription factors displayed tissue-specific expression patterns, with significant differences in expression levels across different tissues. StbHLH003, StbHLH041, and StbHLH068 were differentially expressed under the combined induction of ABA, IAA, GA, MeJA, and SA. All candidate genes exhibited differential responses to drought, NaCl, and mannitol. Expression pattern analysis after re-watering following drought identified several candidate genes, among which StbHLH025 showed high expression levels in leaves and was induced by PEG6000, NaCl, and mannitol, participating in relevant stress responses (Figure 8). These comprehensive analyses have yielded valuable insights into the characteristics of the potato bHLH gene family and have established a robust research platform for further exploration of the specific roles these genes play in the stress tolerance and developmental regulation of potatoes.