Molecular Characterization and Expression of CmobHLH Genes in Pumpkin

: The transcription factor bHLH gene family plays fundamental roles in plant development and mitigating diverse biotic and abiotic stresses. However, the information of bHLH genes in pumpkin ( Cucurbita moschata ) is still unknown. In this current study, 222 CmobHLH genes were identiﬁed and mapped onto different chromosomes through bioinformatics analysis in pumpkin. CmobHLH and AtbHLH proteins could be classiﬁed into 19 subfamilies according to the phylogenetic tree. CmobHLH proteins within the same subfamily had similar motif composition and gene structures. Gene ontology (GO), cis -regulatory elements (CREs) and protein–protein interaction analyses suggested the potential regulatory roles of CmobHLH genes during the plant development process and abiotic stresses response in pumpkin. Tissue expression patterns based on transcriptome data demonstrated that CmobHLH genes were involved in pumpkin development process, and they had unique functions in different tissues. The expression patterns of ﬁve selected CmobHLH genes after exposure to abiotic stresses showed that the CmobHLH genes played varied roles in the stress responses of pumpkin to NaCl, waterlogging, cold, ABA and drought. In brief, these ﬁndings offer important information for further functional research of CmobHLH genes and resistance breeding in pumpkin.


Introduction
The bHLH gene family is one of the most extensive transcription factor (TF) families found in plants, usually classified into 15-26 subfamilies [1]. bHLH family genes play crucial roles in controlling plant development and tolerating diverse environmental stresses through regulating the expression of downstream target genes [2][3][4]. bHLH family proteins are composed of two functional regions: the basic region located in the N-terminus and the HLH region in the C-terminus [5,6]. The basic region, which has 15-20 amino acids (aa), is primarily involved in binding of TFs through binding to the cis-regulatory elements (CREs) [1,7]. The HLH domain has 40-50 aa and is composed of two α-helices connected by a loop, which can promote protein interactions and form homodimers or heterodimers to induce a range of diverse activities [8][9][10]. Identification of bHLH family genes was initially recorded in maize (Zea mays L.) [11]. Thereafter, an increasing number of bHLH genes were widely characterized in numerous plant species, such as Arabidopsis [12], rice [13], tomato [14], poplar [1], apple [15], eggplant [16], grass pea [17], persimmon [18], sweet wormwood [19] and Ipomoea aquatica [20].
A number of evidence has shown that bHLH family genes are involved in plant development processes [21,22]. For example, the bHLH TF SPATULA (SPT) inhibited root growth and size through limiting cell proliferation and expansion in Arabidopsis [23]. EAT1, a rice bHLH TF regulated programmed cell death (PCD) in tapetal cells [24]. Overexpression of AtbHLH68 and AtbHLH112 suppressed lateral root development in Arabidopsis seedlings [25,26]. Some bHLH genes related to fruit development have also been found. Eleven SlbHLH genes have been reported to participate in young fruit development and ripening in tomato [27]. In melon, CmbHLH32 exhibited higher expression in the process of early fruit development, and overexpression of CmbHLH32 led to early fruit ripening [28]. The MYB-bHLH-WD40 complex was positively associated with anthocyanin accumulation and blueberry fruit color development [29]. Furthermore, bHLH family genes significantly strengthened salinity [30], drought [31,32] and cold [33] tolerance. In grapes, expression of the VvICE1a and VvICE1b genes, two bHLH family genes, improved the stresses tolerance of salinity, drought and cold in transgenic Arabidopsis [34]. In addition, bHLH family genes were involved in the regulation of iron homeostasis. In maize, ZmbHLH105 significantly decreased Mn buildup through suppressing the expression of Mn/Fe-regulated transporter genes [35]. In rice, OsbHLH133 was an important regulator of Fe distribution in the roots and shoots [36]. According to the above reports, bHLH family genes would be great candidate genes for improving stress tolerance and resistance breeding in plants.
Pumpkin (Cucurbita moschata) is cultivated and consumed around the world with a very high nutritional and health care value [37]. However, diverse environmental stresses, such as salinity, cold and drought severely affect the processes of growth and development in pumpkin. Although some bHLH genes have been elaborated in plants, and overexpression of CmbHLH87 gene in tobacco increased the resistance to powdery mildew [38], there is no report of CmobHLH genes in resistance to abiotic stresses in pumpkin. In this current study, the CmobHLH gene family was identified and characterized in pumpkin through bioinformatics analysis. Furthermore, we investigated the gene transcription patterns under different tissues and different abiotic stresses, which gave crucial insights for further understanding of the functions of CmobHLH genes and resistance breeding in pumpkin.

Phylogenetic Relationship, Gene Structure and Protein-Protein Interaction Networks of CmobHLH Proteins
Multiple sequence alignments of CmobHLH proteins were conducted using ClustalW software. The phylogenetic tree was constructed using the neighbor-joining (NJ) method with MEGA 7.0 (the Bootstrap = 1000 and other parameters were set to default values) [45]. The online tool Interactive Tree Of Life (iTOL) (https://itol.embl.de/, accessed on 14 February 2023) [46] was used to modify and annotate the generated phylogenetic tree. The syntenic analysis of bHLH genes in pumpkin and Arabidopsis was performed using TBtools [47]. The MEME online tool (https://meme-suite.org/meme/tools/meme, accessed on 14 February 2023) [48] was used for the identification of the conserved motifs, with the default parameters and a maximum number of motifs to 10. The gene structures of the CmobHLH genes were exhibited with TBtools. GO analysis was performed using the GO enrichment tool of CuGenDB, and Gene Ontology [49] was used as an additional validation tool. The 2000 bp upstream promoter sequences of 222 CmobHLH genes were retrieved using TBtools and submitted to the online PlantCARE database (https://bioinformatics. psb.ugent.be/webtools/plantcare/html/, accessed on 14 February 2023) [50] to predict the CREs. Protein-protein interaction networks among CmobHLH proteins were predicted with STRING tool (https://cn.string-db.org/, accessed on 14 February 2023) [51].

Plant Materials, Growth Conditions and Treatments
A six-generation-inbred line of 'Hantailang' was used as the pumpkin material in this study. Pumpkin seeds were germinated at 37 • C for two days, and the germinated seeds were sown in plug trays and grown in the growth chamber under 28 • C with 12 h light and 12 h dark. Pumpkin seedlings at two-leaf stage were treated with different abiotic stresses. For waterlogging, water 2 cm was maintained above the soil surface. For cold, pumpkin seedlings were cultivated at 15 • C day/5 • C night. For ABA and drought stresses, pumpkin seedings were dipped in Hoagland nutrient solution containing 100 µmol ABA and 10% PEG 6000, respectively. Pumpkin leaves were collected on the 10th day after treatment for gene expression analysis.

Gene Expression Analysis
The transcriptional profiles of CmobHLH genes in different tissues were obtained from the RNA-seq data (PRJNA385310) of interspecific F1 hybrid 'Shintosa'. The transcriptional profiles of CmobHLH genes under 75 mmol/L NaCl stress for 24 h were obtained from the RNA-seq data (PRJNA437579) of the root apexes of (Cucurbita maxima × Cucurbita moschata) cv. 'Chaojiquanwang'. In this study, the count of reads was normalized to fragments per kilobase of transcript per million fragments (FPKM) of CmobHLH genes. An expression heatmap was generated with log 2 FPKM values. Expression patterns of 5 CmobHLH genes under waterlogging, cold, ABA and drought stresses were performed using qRT-PCR. For this purpose, total RNA from pumpkin leaves of various treatments was extracted using OminiPlant RNA Kit (CWBIO, Suzhou, China), and PrimeScript™ RT reagent Kit with gDNA Eraser (Takara, Dalian, China) was used to synthesize the first chain cDNA. To demonstrate the expression patterns under diverse stresses, qRT-PCR was performed using TB Green ® Premix Ex Taq™ II (TaKaRa, Dalian, China). Each experiment included three biological replicates, following the manufacturer's protocol. Supplementary Table S1 contains the primer sequences of CmobHLH genes and reference gene (CmoActin) used in this investigation. The relative expression levels of CmobHLH genes were quantified using the 2 −(∆∆Ct) method [52].

Identification and Characterization of CmobHLH Family Genes
In total, 222 bHLH genes were recognized in pumpkin and renamed as CmobHLH1 to CmobHLH222 based on their chromosomal locations. Based on pumpkin genomic information, they were unevenly placed onto the twenty chromosomes of pumpkin ( Figure 1). Chromosome 11 contained the maximum number of CmobHLH genes (19), while chromosome 20 had the minimum number of CmobHLH genes (4). In general, 6-14 CmobHLH genes were present on each chromosome. The characterization of these CmobHLH proteins was presented in Supplementary Table S2. The coding sequence (CDS) lengths of CmobHLH genes varied from 228 to 5469 bp, with 75 to 1822 aa, predicted molecular weight (MW) from 9.32 to 202.73 kDa, isoelectric point (pI) from 4.50 to 9.98 and an instability index (II) from 32.25 to 99.87. The majority of CmobHLH proteins were hydrophilic based on their Grand average of hydropathicity (GRAVY) values. The results of subcellular local-ization revealed that 221 of CmobHLH proteins were located in the nucleus, while only CmobHLH136 was in the cell membrane.
CmobHLH genes varied from 228 to 5469 bp, with 75 to 1822 aa, predicted molecular weight (MW) from 9.32 to 202.73 kDa, isoelectric point (pI) from 4.50 to 9.98 and an instability index (II) from 32.25 to 99.87. The majority of CmobHLH proteins were hydrophilic based on their Grand average of hydropathicity (GRAVY) values. The results of subcellular localization revealed that 221 of CmobHLH proteins were located in the nucleus, while only CmobHLH136 was in the cell membrane.

Phylogenetic Tree and Synteny Analysis of the CmobHLH Proteins
To classify the evolutionary relationships of CmobHLH proteins, the phylogenetic tree was constructed based on 165 AtbHLH and 222 CmobHLH protein sequences. As shown in Figure 2, 387 bHLH proteins were classified into 19 subfamilies. Among them, subfamily 19 comprised the maximum number of CmobHLH proteins (30), followed the subfamily 6 (21), while subfamily 13 contained the minimum number (3). Moreover, CmobHLH proteins and AtbHLH proteins were present in every subfamily; however, the number varied in every subfamily. To further elucidate the synteny of bHLH genes, the synteny map between pumpkin and Arabidopsis was generated. The result showed that 138 CmobHLH genes were determined to be collinear with 103 AtbHLH genes, and 174 orthologous pairs were discovered ( Figure 3).

Phylogenetic Tree and Synteny Analysis of the CmobHLH Proteins
To classify the evolutionary relationships of CmobHLH proteins, the phylogenetic tree was constructed based on 165 AtbHLH and 222 CmobHLH protein sequences. As shown in Figure 2, 387 bHLH proteins were classified into 19 subfamilies. Among them, subfamily 19 comprised the maximum number of CmobHLH proteins (30), followed the subfamily 6 (21), while subfamily 13 contained the minimum number (3). Moreover, CmobHLH proteins and AtbHLH proteins were present in every subfamily; however, the number varied in every subfamily. To further elucidate the synteny of bHLH genes, the synteny map between pumpkin and Arabidopsis was generated. The result showed that 138 CmobHLH genes were determined to be collinear with 103 AtbHLH genes, and 174 orthologous pairs were discovered ( Figure 3). Horticulturae 2023, 9, x FOR PEER REVIEW 5 of 16

Conserved Motifs and Gene Structures of CmobHLH Genes
The MEME online program was employed to indicate the conserved motifs in Cmob-HLH proteins. CmobHLH proteins possessed different number of conserved motifs, ranging from 1-9. CmobHLH proteins from the same subfamily had a common motif composition ( Figure 4B). For instance, the CmobHLH proteins in subfamilies 7, 8, 9, 10, 11 and 18 shared motifs 1, 2, 3 and 4, the CmobHLH proteins in subfamily 4 contained motifs 1 and 2, and the CmobHLH proteins in subfamilies 12, 17 and 19 involved motifs 1, 2, 3 and 5. Furthermore, we found that some of the motifs were only distributed in the specific position of the protein sequences. For example, motif 3 was always distributed at the start of the protein sequences in subfamilies 2, 6, 7, 9, 10, 11, 13, 15, 16 and 18, and motif 5 was almost always distributed at the start of the protein sequences in subfamilies 12, 17 and 19. While motif 4 was almost always distributed at the end of the protein sequences in subfamilies 7, 8, 9, 10, 11, 13, 14, 15 and 18, motif 7 was distributed at the end of the protein sequences in the subfamily 2. In addition, the exon-intron structures of CmobHLH genes

Conserved Motifs and Gene Structures of CmobHLH Genes
The MEME online program was employed to indicate the conserved motifs in Cmob-HLH proteins. CmobHLH proteins possessed different number of conserved motifs, ranging from 1-9. CmobHLH proteins from the same subfamily had a common motif composition ( Figure 4B). For instance, the CmobHLH proteins in subfamilies 7, 8, 9, 10, 11 and 18 shared motifs 1, 2, 3 and 4, the CmobHLH proteins in subfamily 4 contained motifs 1 and 2, and the CmobHLH proteins in subfamilies 12, 17 and 19 involved motifs 1, 2, 3 and 5. Furthermore, we found that some of the motifs were only distributed in the specific position of the protein sequences. For example, motif 3 was always distributed at the start of the protein sequences in subfamilies 2, 6, 7, 9, 10, 11, 13, 15, 16 and 18, and motif 5 was almost always distributed at the start of the protein sequences in subfamilies 12, 17 and 19. While motif 4 was almost always distributed at the end of the protein sequences in subfamilies 7, 8, 9, 10, 11, 13, 14, 15 and 18, motif 7 was distributed at the end of the protein sequences in the subfamily 2. In addition, the exon-intron structures of CmobHLH genes

Conserved Motifs and Gene Structures of CmobHLH Genes
The MEME online program was employed to indicate the conserved motifs in CmobHLH proteins. CmobHLH proteins possessed different number of conserved motifs, ranging from 1-9. CmobHLH proteins from the same subfamily had a common motif composition ( Figure 4B). For instance, the CmobHLH proteins in subfamilies 7, 8, 9, 10, 11 and 18 shared motifs 1, 2, 3 and 4, the CmobHLH proteins in subfamily 4 contained motifs 1 and 2, and the CmobHLH proteins in subfamilies 12, 17 and 19 involved motifs 1, 2, 3 and 5. Furthermore, we found that some of the motifs were only distributed in the specific position of the protein sequences. For example, motif 3 was always distributed at the start of the protein sequences in subfamilies 2, 6, 7, 9, 10, 11, 13, 15, 16 and 18, and motif 5 was almost always distributed at the start of the protein sequences in subfamilies 12, 17 and 19. While motif 4 was almost always distributed at the end of the protein sequences in subfamilies 7, 8, 9, 10, 11, 13, 14, 15 and 18, motif 7 was distributed at the end of the protein sequences in the subfamily 2. In addition, the exon-intron structures of CmobHLH genes were also investigated to obtain their gene structure characteristics using TBtools software ( Figure 4C

GO Enrichment and CREs Analysis
To determine the specific functions of CmobHLH genes, 222 CmobHLH genes were annotated with GO enrichment. The findings revealed that the 222 CmobHLH genes were mainly enriched in three categories (Supplementary Table S3). According to the biological process, they were predicted to be involved in the metabolic process, biosynthetic process, gene expression and other processes. Additionally, the majority of CmobHLH genes were annotated to biosynthetic process (146/222). In the cellular component, most of the CmobHLH genes were assigned to the cellular component (174/222). CmobHLH genes were also assigned to the protein complex (40/222), transcription factor complex (39/222) and nucleus (38/222). In the molecular function, most of CmobHLH genes were involved in binding (222/222), protein binding (221/222) and protein dimerization activity (220/222). A part of CmobHLH genes were involved in nucleic acid binding transcription factor activity (39/222), transcription factor activity, sequence-specific DNA binding (39/222) and DNA binding (35/222). A few CmobHLH genes were involved in cytoplasm amino acid binding, carboxylic acid binding and organic acid binding.
To further investigate the potential functions of CmobHLH genes, the CREs in the promoter sequence of each CmobHLH gene were predicted. As shown in Supplementary  Table S4, it was discovered that 222 CmobHLH genes contained a plethora of light response elements, such as Box 4, G-box, GT1-motif, I-box and MRE. Plant growth and development CREs were also identified, including CAT-box, CCGTCC-box, GCN4_motif, O2-site and circadian, etc. CREs related to biotic and abiotic stress elements, including ARE, WUNmotif, LTR, STRE and MBS were widely present in the promoter region. Furthermore, phytohormone response elements, such as ABRE, TGA-element, P-box, CGTCA-motif and ERE were found. The different types of CREs presenting in CmobHLH genes indicated functional diversity and complexity.

Tissue Expression of CmobHLH Genes
We used RNA-seq data to examine the transcriptional levels of CmobHLH genes the roots, stems, leaves and fruits to ascertain their roles ( Figure 6). In the four tissu studied, 222 CmobHLH genes were expressed at varying abundance levels. Three obvio transcriptional clusters were shown, with the higher abundance in cluster I and the low in cluster III. Furthermore, we found that the expression levels of CmobHLH genes in clu ter I were generally high in the four tissues, while CmobHLH55 and CmobHLH179 we the highest in the fruits. A majority of CmobHLH genes in cluster II exhibited higher e pression in the stems and roots. However, the expression levels of CmobHLHs in clust III were generally lower in all four tissues. The various expression profiles of CmobHL genes in different tissues suggested the different functions in the growth and developme of pumpkin.

Tissue Expression of CmobHLH Genes
We used RNA-seq data to examine the transcriptional levels of CmobHLH genes in the roots, stems, leaves and fruits to ascertain their roles ( Figure 6). In the four tissues studied, 222 CmobHLH genes were expressed at varying abundance levels. Three obvious transcriptional clusters were shown, with the higher abundance in cluster I and the lower in cluster III. Furthermore, we found that the expression levels of CmobHLH genes in cluster I were generally high in the four tissues, while CmobHLH55 and CmobHLH179 were the highest in the fruits. A majority of CmobHLH genes in cluster II exhibited higher expression in the stems and roots. However, the expression levels of CmobHLHs in cluster III were generally lower in all four tissues. The various expression profiles of CmobHLH genes in different tissues suggested the different functions in the growth and development of pumpkin.

Expression Analysis of CmobHLH Genes under Abiotic Stresses
CREs analysis indicated that CmobHLH genes could be associated with stress response. To further explore possible functions under abiotic stresses, expression profiles of five selected CmobHLH genes were detected with the treatments of salinity, waterlogging, cold, ABA and drought. As shown in Figure 7, transcriptional levels of all five CmobHLH genes were up-regulated after 24 h of 75 mmol/L NaCl stress compared with the normal condition. Furthermore, the expression profiles of CmobHLH genes under waterlogging, cold, ABA and drought stresses were analyzed using qRT-PCR. The results demonstrated that five CmobHLH genes exhibited a significantly up-regulated expression under cold, ABA and drought stresses after 10 days, in contrast with the normal condition ( Figure 8). However, CmobHLH25, CmobHLH46, CmobHLH81 and CmobHLH167 showed higher expression levels by 1.64-, 1.82-, 1.35-, 1.17-fold under waterlogging stress, respectively, while the expression of CmobHLH98 was down-regulated. From the above results, we learned that the five selected CmobHLH genes could respond to NaCl, waterlogging, cold, ABA and drought stress in pumpkin.

Expression Analysis of CmobHLH Genes under Abiotic Stresses
CREs analysis indicated that CmobHLH genes could be associated with stress response. To further explore possible functions under abiotic stresses, expression profiles of five selected CmobHLH genes were detected with the treatments of salinity, waterlogging, cold, ABA and drought. As shown in Figure 7, transcriptional levels of all five CmobHLH genes were up-regulated after 24 h of 75 mmol/L NaCl stress compared with the normal condition. Furthermore, the expression profiles of CmobHLH genes under waterlogging, cold, ABA and drought stresses were analyzed using qRT-PCR. The results demonstrated that five CmobHLH genes exhibited a significantly up-regulated expression under cold, ABA and drought stresses after 10 days, in contrast with the normal condition ( Figure 8). However, CmobHLH25, CmobHLH46, CmobHLH81 and CmobHLH167 showed higher expression levels by 1.64-, 1.82-, 1.35-, 1.17-fold under waterlogging stress, respectively, while the expression of CmobHLH98 was down-regulated. From the above results, we learned that the five selected CmobHLH genes could respond to NaCl, waterlogging, cold, ABA and drought stress in pumpkin.

Expression Analysis of CmobHLH Genes under Abiotic Stresses
CREs analysis indicated that CmobHLH genes could be associated with stress response. To further explore possible functions under abiotic stresses, expression profiles of five selected CmobHLH genes were detected with the treatments of salinity, waterlogging, cold, ABA and drought. As shown in Figure 7, transcriptional levels of all five CmobHLH genes were up-regulated after 24 h of 75 mmol/L NaCl stress compared with the normal condition. Furthermore, the expression profiles of CmobHLH genes under waterlogging, cold, ABA and drought stresses were analyzed using qRT-PCR. The results demonstrated that five CmobHLH genes exhibited a significantly up-regulated expression under cold, ABA and drought stresses after 10 days, in contrast with the normal condition ( Figure 8). However, CmobHLH25, CmobHLH46, CmobHLH81 and CmobHLH167 showed higher expression levels by 1.64-, 1.82-, 1.35-, 1.17-fold under waterlogging stress, respectively, while the expression of CmobHLH98 was down-regulated. From the above results, we learned that the five selected CmobHLH genes could respond to NaCl, waterlogging, cold, ABA and drought stress in pumpkin.

Discussion
bHLH TFs have been identified and characterized in many plants. However, information about bHLH genes in resistance to abiotic stresses in pumpkin is missing. In this study, we identified and characterized 222 CmobHLH genes in pumpkin, which were further divided into 19 subfamilies based on their alignment with AtbHLH proteins. Furthermore, CmobHLH proteins within the same subfamily were discovered to have comparable motif composition and gene structures, highlighting the closer evolutionary relationship of CmobHLH proteins in the same subfamily. Ke et al. [61] reported that genes from the same subfamily shared the common evolutionary origin and similar physiological functions.
The CREs analysis in the promoter regions offers a theoretical foundation for investigating the physiological functions of CmobHLH genes. bHLH genes have been reported to participate in phytohormone signal crosstalk, which plays crucial roles in plant development and response to various abiotic stresses [62][63][64][65][66][67]. Previous studies have also shown the expression profiles of bHLH genes in response to salicylic acid (SA), 6-benzylaminopurine (6-BA) [68] and jasmonic acid (JA) [69]. In this study, there were a series of CREs in the promoters of CmobHLH genes, including light, plant growth and development, abiotic stresses and plant phytohormones, suggesting that these genes serve as regulators that are widely involved in growth and development, as well as various stress responses in pumpkin. In addition, GO annotation and protein-protein interaction networks also indicated the important roles of CmobHLH genes in pumpkin development and stress response.
Transcriptome data in the different tissues, as well as protein-protein interaction networks analysis, might aid in predicting the potential roles of the studied genes. In Prunus mume, many PmbHLH genes were specifically expressed in the roots, implying that they might be connected to root development [57]. In fig, a large number of FcbHLH genes Figure 8. Expression levels of CmobHLH genes in response to waterlogging, cold, ABA and drought stresses after 10 days. The error bars are denoted as the means ± SEs. Asterisks indicate significant differences at * p < 0.05, ** p < 0.01, *** p < 0.001 and **** p < 0.0001.

Discussion
bHLH TFs have been identified and characterized in many plants. However, information about bHLH genes in resistance to abiotic stresses in pumpkin is missing. In this study, we identified and characterized 222 CmobHLH genes in pumpkin, which were further divided into 19 subfamilies based on their alignment with AtbHLH proteins. Furthermore, CmobHLH proteins within the same subfamily were discovered to have comparable motif composition and gene structures, highlighting the closer evolutionary relationship of CmobHLH proteins in the same subfamily. Ke et al. [61] reported that genes from the same subfamily shared the common evolutionary origin and similar physiological functions.
The CREs analysis in the promoter regions offers a theoretical foundation for investigating the physiological functions of CmobHLH genes. bHLH genes have been reported to participate in phytohormone signal crosstalk, which plays crucial roles in plant development and response to various abiotic stresses [62][63][64][65][66][67]. Previous studies have also shown the expression profiles of bHLH genes in response to salicylic acid (SA), 6-benzylaminopurine (6-BA) [68] and jasmonic acid (JA) [69]. In this study, there were a series of CREs in the promoters of CmobHLH genes, including light, plant growth and development, abiotic stresses and plant phytohormones, suggesting that these genes serve as regulators that are widely involved in growth and development, as well as various stress responses in pumpkin. In addition, GO annotation and protein-protein interaction networks also indicated the important roles of CmobHLH genes in pumpkin development and stress response.
Transcriptome data in the different tissues, as well as protein-protein interaction networks analysis, might aid in predicting the potential roles of the studied genes. In Prunus mume, many PmbHLH genes were specifically expressed in the roots, implying that they might be connected to root development [57]. In fig, a large number of FcbHLH genes exhibited widespread expression in the fruits, indicating that FcbHLH genes play a significant role in fig fruit development [70]. In this current study, 222 CmobHLH genes were divided into three clusters according to their abundance levels, and the expression profiles of CmobHLH genes varied in different tissues. For example, CmobHLH55 and CmobHLH179 were the highest in the fruits, and the majority of CmobHLH genes in cluster II exhibited higher expression in the stems and roots, which suggested the unique functions of CmobHLH genes in pumpkin development. Additionally, bHLH TFs have also been discovered to play an essential role in the process of plant response to abiotic stresses [71]. For instance, in grass pea, most of LsbHLH genes showed higher expression levels under 75 mM NaCl treatment [17]. In cucumber, the expression profile of CsbHLH032 was downregulated after 4 • C treatment [72]. In Hibiscus hamabo, HhbHLH20 displayed significantly lower expression in response to ABA stress [73]. In Mongolian oak, the expression levels of QmbHLH81 and QmbHLH30 first increased significantly and then decreased remarkably with PEG 6000 treatment [74]. In sorghum, SbbHLH045 showed significant up-regulation under flooding stress [75]. This current study showed that CmobHLH25, CmobHLH46, CmobHLH81, CmobHLH98 and CmobHLH167 exhibited an increased expression after 24 h of NaCl stress and 10 days of cold, ABA and drought, indicating that CmobHLH genes might play the vital roles in NaCl, cold, ABA and drought stresses. Additionally, CmobHLH25 and CmobHLH46 were significantly expressed after 10 days of waterlogging stress, indicating an opposite pattern for CmobHLH25 and CmobHLH46 in response to waterlogging stress. Our results were aligned with previous reports on Andrographis paniculate [68], banana [76] and foxtail millet [77]. In summary, CmobHLH genes might mediate the stress response of pumpkin to NaCl, cold, ABA, drought and waterlogging, but the activities of individual CmobHLH genes were different with the different stress. Further research is required to comprehend the functions of CmobHLH genes through performing a gain-of-function or loss-of-function assay.

Conclusions
In general, we identified 222 CmobHLH genes in pumpkin. Furthermore, a systematic investigation was carried out, including chromosomal localization, protein properties, phylogenetic tree, synteny analysis, conserved motifs, gene structures, GO enrichment, CREs and protein-protein interaction networks. Gene expression patterns in different tissues and response to abiotic stresses were examined in pumpkin. Our results present a systematic understanding of the characterization and demonstrated the potential roles in development and stress response in pumpkin, which would provide foundation for further studies on the functions and regulatory mechanisms of CmobHLH genes in specific environments.