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Genome-wide characterization and expression analysis of the bHLH gene family in response to abiotic stresses in Zingiber officinale Roscoe

BMC Genomics volume 26, Article number: 143 (2025) Cite this article

Abstract

Background

The basic helix-loop-helix (bHLH) transcription factors play important physiological functions in the processes of plant growth, development, and response to abiotic stresses. However, a comprehensive genome-scale study of the ginger bHLH gene family has not been documented.

Results

In this study, 142 ZobHLH genes were identified in the ginger genome. Using Arabidopsis bHLH proteins as a reference, ZobHLH genes were classified into 15 subfamilies and unevenly distributed on 11 chromosomes of ginger. Sequence characterization, multiple sequence alignment, phylogenetic analysis, conserved protein motifs and exon-intron distribution patterns were conducted to further analyze the evolutionary relationships among these ZobHLH proteins. The results of the duplicated event analysis demonstrated that the pivotal role of segment duplication in promoting the expansion of the ZobHLH gene family. Additionally, analysis of cis-regulatory elements as well as protein interaction networks indicated the potential involvement of ginger ZobHLH family proteins in plant growth and development, and response to adversity stress. RNA-seq and RT-qPCR results showed that ZobHLH083 and ZobHLH108 play key roles in response to salt stress and waterlogging stress, respectively.

Conclusion

In this study, we systematically analyzed the characteristics of ZobHLH proteins in ginger, discovering that these genes play critical roles in ginger rhizome expansion and response to salt and waterlogging stresses. The present study provides a theoretical foundation for the further research on ZobHLHs and will help to explore the functional properties of ZobHLH genes.

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Background

Adverse factors such as heavy metals, drought, high salt, or extreme temperatures, which significantly disrupt plant normal physiological activities, reducing production and quality [1]. However, plants have evolved complex signal sensing and defense systems when exposed to adversity. During this process, transcription factors (TFs) play critical roles and mainly function to regulate relative genes expression and then modify plant growth, physiology and metabolic pathways, and then enhance the resistance to abiotic stresses [2]. For example, MYB TFs can bind to MYB-binding elements in the promoter regions of various functional genes to activate the expression level of stress-responsive genes and then initiate plant response to adversity [3]. The bHLH family is one of the largest TF families in plants and plays an important role in plant response to abiotic stresses including waterlogging and high salt stress [4].

The bHLH TFs were first identified in animals and later in the majority of eukaryotes, deriving its name from the presence of the bHLH (basic Helix-Loop-Helix) domain, which spans approximately 60 amino acids [5, 6]. The N-terminus features an α-helix within the basic structural domain, comprising approximately 15–20 amino acids and enriched with basic residues binding to the cis-elements. The HLH region is located to the C-terminal, containing two α-helices connected by a relatively poorly conserved loop, and relies on the interaction of hydrophobic amino acids to form homo- or heterodimers [7, 8]. There are 15 to 32 subfamilies according to the taxonomy of bHLH proteins [9, 10]. Most bHLH TFs are crucial for plant growth and metabolism, influencing secondary metabolism, light morphogenesis and signal transduction [11]. For example, maize anthocyanins are regulated by the highly conserved MYB-bHLH-WD40 transcriptional regulatory complex [12]. In Fragaria ananassa, the genes FabHLH25, FabHLH29, FabHLH80, and FabHLH98 are involved in fruit anthocyanins production and hormone signal transduction [13]. The TF AabHLH112 enhances the production of sesquiterpenes in Artemisia annua [14]. Conversely, silencing of the genes SmMYC2a and SmMYC2b results in reduced accumulation of tanshinone and phenolic acids [15]. In addition, bHLH TFs play important roles in plant stress response. The pepper gene CabHLH035 promotes salt tolerance by regulating ion homeostasis and proline biosynthesis [16]. Overexpressing SlbHLH22 and SlbHLH96 in tomato enhances drought and salt tolerance [17, 18]. OsbHLH057 activates the expression of the Os2H16 gene, thereby promoting disease resistance and drought tolerance in rice [19]. Moreover, bHLH proteins regulate tryptophan biosynthesis and various hormones, significantly contributing to hormone-mediated regulatory networks [10, 20]. In Arabidopsis, bHLH3, bHLH13, bHLH14 and bHLH17 function as transcriptional repressors affecting responses to jasmonic acid (JA) [21]. In sweet sorghum, SbbHLH85 plays a crucial role in root development and mediates the response to salt stress by regulating abscisic acid (ABA) and auxin signaling pathways [22]. Therefore, bHLH TFs are integral to plant development and stress response. However, studies on the function and physicochemical properties of the ZobHLH genes in Zingiber officinale Roscoe are lacking.

Ginger, widely utilized for its medicinal and culinary properties, is globally cultivated primarily for its underground rhizome [23]. Ginger has great economic value, but it is susceptible to various detrimental factors, such as salt [24], drought [25], waterlogging [26], and high temperature [27]. Recent studies have shown that salinity causes a decrease in photosynthetic capacity of ginger, and plant dwarfing [28]. Waterlogging inhibits the root vitality and hampers its growth and development [29]. Both of them lead to a decrease in the accumulation of minerals in ginger, which in turn cause a significant reduction in its yield. Uncovering the mechanisms underlying growth, development and tolerance to abiotic stresses can facilitate the selection and breeding of resilient and high-yielding ginger cultivars. Recent advancements in sequencing technology and the completion of the ginger genome sequence have enabled the identification of gene families involved in stress responses on a genome-wide scale [30]. This study employs bioinformatics to identify ZobHLH family genes in ginger, analyzing their physicochemical properties, gene structure, conserved motifs, phylogenetic relationships and gene expression patterns, which will elucidate the roles of ZobHLHs in abiotic stress responses. These results will provide a global perspective for the study of evolution and functional characterization of the ZobHLHs in ginger, as well as further research on the function of ZobHLHs in growth and development and the mechanism of stress resistance, and also provide a theoretical reference for the breeding of stress-tolerant ginger varieties.

Materials and methods

Identification of the ZobHLH gene family

The ginger bHLH family members were identified by Hidden Markov Modelling (HMMER) and the ginger genome has been published online [30]. The HMMER profile for HLH (PF00010) was downloaded from the Pfam database (https://pfam.xfam.org/), and the candidate genes were obtained from the ginger genome using HMMER software with an e-value cutoff set to 10−5. The protein sequences of the identified genes were subsequently analyzed for redundant and structurally incomplete domain sequences using NCBI_CDD (https://www.ncbi.nlm.nih.gov/Structure) and SMART (http://smart.embl-heidelberg.de/). The genes were then renamed sequentially based on their chromosomal positions, and the physicochemical properties of ZobHLH proteins predicted by ExPASy (https://web.expasy.org/protparam/).

Sequence alignment and phylogenetic relationship, gene structure, and conserved motif analyses of the ZobHLHs

Multiple sequence alignment of ZobHLH protein sequences was performed with ClustalW2, and phylogenetic trees were constructed using MEGA 11.0 software by Neighbor-joining (NJ) with 1000 bootstrap replicates [31]. The bHLH protein sequences of Arabidopsis were downloaded from the PlantTFDB website (https://planttfdb.gao-lab.org/). Conserved structural domains were identified using the WEB LOGO tool (http://weblogo.berkeley.edu/logo.cgi) to generate sequence logos. The exon-intron structure of ZobHLHs was obtained from the gene annotation files of the ginger genome and visualized by the online software gene structure display server (http://gsds.cbi.pku.edu.cn/). The conserved motifs of ZobHLH proteins were identified by MEME v5.1.1 (http://meme-suite.org/tools/meme), allowing for a maximum of 20 and optimal motif widths ranging from 6 to 50.

Chromosomal distribution, gene duplication and synteny of the ZobHLHs

The genomic location of ZobHLHs was mapped to their respective chromosomes using an annotation file from the ginger genome. Gene duplication events within ZobHLHs were analyzed using the Multiple Co-linear Scanning Toolkit (MCScanX) with default parameters [32]. Comparative collinearity analyses between ginger and other species (including Arabidopsis, rice, banana, maize, tomato) were detected and visualized using TBtools v1.089 [33]. Genome sequences and annotation files of Arabidopsis, rice, banana, maize, and tomato were downloaded from EnsemblPlants (https://plants.ensembl.org/index.html). Ka/Ks analyses were performed to evaluate selection pressure by the online software PAL2NAL (https://www.bork.embl.de/pal2nal/).

Analyses of cis-regulatory elements and prediction of the protein-protein interaction network of the ZobHLHs

Sequence located 2000 bp (base pairs) upstream of the ZobHLHs genes were extracted and analyzed for cis-acting elements using PlantCARE (https://bioinformatics.psb.ugent.be/webtools/plantcare/html/). Furthermore, the STRING database (https://string-db.org/) was employed for the interaction networks and a confidence threshold was set to 0.4. The network was visualized by Cytoscape software (v3.10.1).

GO (gene ontology) functional annotation and enrichment analysis of ZobHLHs

The ZobHLH protein sequences were annotated using the Blast2GO program to assign the GO terms (https://amigo.geneontology.org/amigo/term/), and the e-value for GO analysis was 1.0e-6.

Plant materials and expression analysis of ZobHLHs by RNA‑seq and RT‑qPCR

The ginger species utilized in this study was ‘Zhugen’ ginger. Initially, the gingers were germinated under controlled conditions with a 24 h dark photoperiod at 26 °C and 70% relative humidity. Upon budding, healthy ginger buds were selected and cultivated in the field, and samples were collected at five stages of rhizome expansion (R1 ~ Rh5), respectively [34]. Simultaneously, another ginger buds were transplanted into pots with a diameter of 20 cm and grown in mixed substrate including garden soil, cocopeat, and perlite in a ratio of 6:3:1 under conditions of 26 °C, approximately 10,000 lx light intensity, 70–80% humidity, and 12-hour light-dark photoperiod.

After two months of routine cultivation and management, ginger seedlings with uniform and robust growth were selected for stress treatments. For salt stress, the plants were watered with 20 g L−1 of NaCl solution for three consecutive days. For waterlogging stress, the double-jacketed pot method [29] was employed to maintain soil at a supersaturated level, with periodic rehydration until the water surface was 3 cm above the soil surface. Samples including leaves and roots under salt and waterlogging stress treatments for 3 days were collected, respectively, and then rapidly snap-freezing in liquid nitrogen for RNA-seq analysis and RT-qPCR analyses.

Total RNA was extracted according to the instruction of FastPure Universal Plant Total RNA Isolation Kit (Vazyme), and cDNA synthesis was performed based on HiScript II Q RT SuperMix for qPCR (+ gDNA wiper) (Vazyme). RT-qPCR analysis was conducted using Taq Pro Universal SYBR qPCR Master Mix (Vazyme) on a CFX Connect Fluorescent Quantitative PCR Detection System (Bio-Rad Laboratories). Each 20 µL reaction mixture contained 10 µL Taq Pro Universal SYBR qPCR Master Mix, 0.8 µL primer (10 µM Forward and Reverse), 6.7 µLddH2O, and 2.5 µL cDNA template. The amplification progarm for RT-qPCR run as follows: initial denaturation at 95 °C for 3 min, followed by 44 cycles of 95 °C for 30 s and 60 °C for 30 s, with a melt curve analysis from 60 to 95 °C. DEGs (Differently expressed genes) with FPKM (fragments per kilobase of transcript per million fragments mapped) values greater than 15 and log2(FC) greater than 1.5 were selected for quantification of leaves and roots under salt and waterlogging stress, and a total of 17 genes were screened, namely, ZobHLH003/018/020/032/043/065/083/086/088/095/108/111/122/126/128/131/142. The reference gene RBP [35] was used for normalization, and each experiment included three biological replicates. Relative expression levels of ZobHLHs were calculated by 2−ΔΔCT method [36]. Primer information for RT-qPCR were were listed in Supplementary Table S1.

Subcellular location analyses

Predictions for subcellular localization of ZobHLH proteins were conducted using WOLF PSORT69 (https://www.genscript.com/psort.html). To further determine the location of the ginger ZobHLH genes, ZobHLH083 and ZobHLH108 which did not contain a stop codon, were amplified by PCR with a pair of specific primers (Supplementary Table S2), and the entire coding regions of ZobHLH083 and ZobHLH108 were constructed into the pCAMBIA1302 vector which contained a GFP protein under the control of CaMV 35 S, respectively. Subsequently, the recombinant plastids were transferred into Agrobacterium tumefaciens GV3101, then injected into the epidermal leaves of tobacco for 48 h. The GFP signal can be observed under a laser confocal microscope (Olympus FV 1200).

Results

Identification and physicochemical properties of the ZobHLHs

After removing redundancy, a total of 142 ZobHLH protein sequences containing bHLH conserved structure domains were identified. All the ZobHLH protein sequences were listed in Supplementary Table S3. ZobHLH genes were mapped to 11 chromosomes and sequentially renamed as ZobHLH001 ~ ZobHLH142 based on their chromosomes position (Supplementary Table S4).

The physicochemical properties of ZobHLH proteins varied widely (Supplementary Table S5). According to the prediction of ExPASy, these proteins ranged in length from 95 to 1134 amino acids, with molecular masses between 10615.09 and 126549.30 Da, and theoretical isoelectric points (pI) from 4.80 to 9.87, Among them, 96 ZobHLH proteins were acidic (pI< 7), and 46 were classified as basic (pI> 7). The protein with the lowest instability index was ZobHLH020 (42.44), while the highest was ZobHLH079 (78.86). All the proteins of instability indices were greater than 40, indicating ZobHLH belong to unstable proteins. The aliphatic index ranged from 53.75 (ZobHLH123) to 96.28 (ZobHLH034). The grand average of hydropathicity (GRAVY) was negative, showing that ZobHLHs were hydrophilicity.(Supplementary Table S4). Subcellular localization predictions revealed that 123 ZobHLH proteins were localized in the nucleus, six in the chloroplast, five in the cytoplasm, three in the mitochondrion, two in both the peroxisome and plasma membrane, and one in the endoplasmic reticulum (Supplementary Table S6).

Multiple sequence alignment, phylogenetic analysis, and classification of ZobHLHs

According tomultiple sequence comparison, all the ZobHLHs contain a conserved basic region and two helical regions (Fig. 1). Notably, residues Glu-4, Arg-5, Arg-9, and Arg-10 in the basic region, Leu-20 and Pro-24 in helix 1, and Asp-58, Ala-60, Leu-66, Leu-76 in helix 2 show high conservation (> 75%). Remarkably, residues Arg-9, Arg-10, Leu-20, Pro-26, and Leu-76 demonstrate a consistency level exceeding 95%, highlighting their critical roles in ZobHLHs function.

Fig. 1
figure 1

Multiple sequence comparison of structural domains of ZobHLH proteins. A Sequence logo for the ZobHLH domains. The overall height of each stack represents the conservation of the sequence at that position. B Different color indicates the degree of amino acid identity at each position

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To analyze the evolutionary relationships of ginger ZobHLH gene family, 142 ZobHLH proteins were aligned with 153 A. thaliana bHLH proteins using ClustalW2. As shown in Fig. 2 and 295 bHLH proteins were categorized into 15 distinct subfamilies (A-O), which consistent with the classification results proposed by Wang et al. [37]. ZobHLHs were distributed in all subfamilies, subfamily A contains 23 proteins, while subfamily G includes only one ZobHLH protein (Fig. 2).

Fig. 2
figure 2

Phylogenetic tree of bHLH TFs in ginger and Arabidopsis. Different colors indicate different subfamilies, red stars indicate ginger, skyblue circles represent Arabidopsis

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Gene structure, and conserved motifs of the ZobHLHs

Analysis of conserved motifs among ZobHLH proteins revealed variations both in the type and number. As shown in Fig. 3B, all the ZobHLH proteins contained highly conserved Motif-1 and Motif-2. While Motif-15 existed exclusively in 11 members of subfamily N. ZobHLHs in same subfamily usually have similar motif compositions. For example, genes in subfamily M typically contained Motif-1, Motif-2, and Motif-3, while members of the subfamily O contained Motif-14 in addition to the motifs found in subfamily M. Interestingly, some conserved motifs were found to occupy specific positions in the protein sequence. Motif-9 and Motif-10 were invariably located at the N-terminus, while Motif-4, Motif-8, Motif-5, Motif-13 and Motif-15 were consistently located at the C-terminus. Further analysis indicates that all the proteins contain the bHLH characteristic domain (Fig. 3C), some contain additional domains, such as a reticulon domain in ZobHLH039, and a ZnF_C2HC structure domain in ZobHLH062. For the gene structure, we found that 142 ZobHLH genes exhibit exon counts ranging from one to 11 (Fig. 3D). Except from 13 ZobHLHs that lack introns, the CDSs of the remaining genes are interrupted by introns, and the intron numbers ranging from one to 10. Notably, eight of the intron-lacking genes belong to subfamily C, while five belong to subfamily I. Despite differences in exon locations, genes in the same subfamily generally exhibit similar exon numbers and distribution patterns.

Fig. 3
figure 3

Conserved motifs, gene structures and phylogenetic relationships of ZobHLHs. A Phylogenetic tree of 142 ZobHLH proteins. B Conserved motifs in ZobHLHs, the conserved motifs are represented in different colors, black lines indicate introns. C Structural domains contained in ZobHLHs, green indicates the bHLH structure domain, yellow indicates the bHLH-MYC_N structure domain, red indicates the reticulon structure domain and dark green indicates the ZnF_C2HC structure domain. D Exon-intron structural domains of the ZobHLHs, yellow boxes indicate exons, green boxes indicate 5‘- and 3’-untranslated regions

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Chromosomal distribution, evolutionary analyses of the ZobHLHs within and between species

As was shown in Fig. 4A, the distribution of ZobHLH genes across the chromosomes was uneven. For example, Chr04 contained eight genes, Chr06 contained 19 genes, and the remaining chromosomes contained between 10 and 17 ZobHLHs. In this study, A tandem duplication of genes (ZobHLH076/077) on Chr06 was detected (Fig. 4A). Additionally, 42 pairs of segment duplication genes were detected, involving 74 ZobHLH genes, in their respective subfamilies (Fig. 4B). To elucidate the evolutionary pressures influencing the bHLH gene family, the ratios of KA/KS for ZobHLH gene pairs were calculated (Supplementary Table S7). The results showed that all the homologous gene pairs have KA/KS values less than 1, suggesting that purifying selection has strongly shaped the evolution of ZobHLH genes.

Fig. 4
figure 4

Chromosomal distribution and segmental duplication collinearity analysis. A Chromosome distributions of ZobHLHs. All chromosomes have the ZobHLH genes and the red arcs connect the tandem duplication genes. B Chromosomal distribution and segmental duplication collinearity map of the ZobHLHs in ginger. Blue font and blue lines indicate segmental duplication gene pairs in the ginger genome

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To further elucidate the evolutionary relationship of the bHLH genes across different species, a collinearity map comparing ginger with six other species was constructed using TBtools (Fig. 5, Supplementary Table S8). The results showed that no collinear genes existed between ginger and Arabidopsis, while seven pairs of colinear genes were identified in tomato. In contrast, 16 pairs of collinear genes were found in rice, 19 pairs in maize, 43 pairs in wheat, and 184 pairs in banana. These results demonstrate that ginger shares a closer evolutionary relationship with monocotyledons compared to dicotyledons.

Fig. 5
figure 5

Synteny analysis of bHLH genes between ginger and five plant species (A. thaliana, Solanum lycopersicum, Oryza sativa, Zea mays, Triticum aestivum, and Musa acuminata). The grey line shows the collinearity of ginger with the other species, while the red line highlights the collinearity bHLH gene pairs

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Cis-element analyses of the ZobHLHs

To determine the type and distribution of cis-acting elements in the ZobHLH promoter, we extracted 2000 bp upstream of the ZobHLHs using PlantCARE. As showed in Fig. 6 and Supplementary Table S9, the predominant cis-elements are associated with hormone response, including auxin, abscisic acid, gibberellins, methyl jasmonate, salicylic acid, et al. Additionally, several cis-elements related to plant growth and development were identified, such as cis-elements involved in endosperm expression, meristem expression, and flavonoid biosynthetic genes regulation. Notably, some cis-elements related to stress responses, including defense and stress responsiveness, like MYB binding site involved in drought-inducibility, wound responses, anoxic specific inducibility, low-temperature responsiveness and other abiotic stress-related components. In Fig. 6B, among these three types of components (hormone, growth and stress responsiveness), the number of components related to abiotic stress was the highest, indicating that ZobHLH genes played important roles in responding to abiotic stress.

Fig. 6
figure 6

Cis-regulatory elements of the ZobHLH promoters. A Distribution of cis-acting elements in the 2000 bp sequence upstream of the 142 ZobHLH genes, with different color blocks indicating different acting elements, and some elements overlapping. B Histogram showing the proportion of various cis-elements, different colors indicate different cis-element types

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Prediction of the ZobHLHs protein-protein interaction network

The prediction of protein-protein interaction revealed significant interactions between the ZobHLH proteins (Fig. 7), which could be categorized into three layers based on their connectivity [23]. The innermost circle, characterized by the highest connectivity (> 15), comprises four members, namely ZobHLH103、ZobHLH126、ZobHLH132 and ZobHLH141. The second circle contains 12 ZobHLHs, and the outermost circle contains 30 ZobHLH proteins with connectivity less than nine. Among them, ZobHLH103 is homologous to Arabidopsis BIM2, ZobHLH126 is homologous to AtbHLH041, ZobHLH132 is homologous to AtbHLH93, and ZobHLH141 is a homologous protein of AtbHLH115. BIM2 is important in brassinosteroid signaling [38], AtbHLH93 is involved in stomatal defense and cell differentiation [39], AtbHLH041 is associated with the signaling of auxin and cytokinin [40], AtbHLH115 plays an important role in iron homeostasis and adversity regulation [41]. Hence, we can speculate that ZobHLH proteins may function in several aspects of plant growth and development, including hormone signaling, cell differentiation, and adversity response.

Fig. 7
figure 7

Interaction network of ZobHLH proteins. Grey line connections indicate predicted interactions between the proteins, and the nodes (red circles in the figure) range from large to small and from dark to light red, indicating a gradual decrease in the number of interacting proteins

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GO analysis of ZobHLHs proteins

To further elucidate the functions of ZobHLHs genes, we conducted gene ontology annotation analysis. The GO annotation of ZobHLH includes biological processes, molecular function and cellular component (Supplementary Table S10). In particular, ZobHLH proteins are involved in a variety of biological processes, including the regulation of biological processes, intracellular metabolic processes, regulation of DNA-templated transcription, anatomical structure development and response to abiotic stimulus. Among the molecular functions, ZobHLH proteins are involved in DNA-binding transcription activity, and DNA binding. In the cellular component, ZobHLH proteins are mainly involved in intracellular membrane-bounded organelle, membrane-bounded organelle.

Expression profiles of the ZobHLHs in ginger at different developmental stages

To analyze the expression profiles of ZobHLHs during ‘Zhugen’ ginger rhizome expansion, we determined transcriptome data from five developmental stages (Supplementary Table S11, Fig. 8A). At the Rh1 stage, 42 ZobHLH genes were significantly increased (FPKM ≥ 5). There are 37, 38, 41, and 57 ZobHLHs upregulated in Rh2, Rh3, Rh4, and Rh5 stages, respectively. Interestingly, 34 genes were highly expressed at all five expansion periods simultaneously, such as ZobHLH065, ZobHLH064, et al.

Fig. 8
figure 8

Expression profiles of the ZobHLHs in ginger at different developmental stages and abiotic stresses. A Heat map of ZobHLH genes expression during five developmental periods of ginger rhizome expansion. B Expression profiles of ZobHLH genes in leaves and roots of ginger under salt stress and waterlogging stress. Log2FPKM normalization was used, and color blocks from blue to red indicate low to high gene expression. C Venn diagram of DEGs up-regulated and down-regulated in leaves and roots under salt and waterlogging stresses

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The expression level of ZobHLHs in ginger under salt and waterlogging treatments

In this study, we performed RNA-seq analysis on different parts of ‘Zhugen’ ginger subjected to salt and waterlogging stresses, respectively (Supplementary Table S12, Fig. 8B). Under salt stress, 48 genes showed significant differential expression, with 28 in leaves (15 up-regulated) and 25 in roots (three up-regulated). Four genes (ZobHLH142, ZobHLH022, ZobHLH139, and ZobHLH077) were differentially expressed in both leaves and roots, among which ZobHLH142 up-regulated in all tissues. Also in leaves, ZobHLH083 was significantly up-regulated and had the highest expression level. After waterlogging treatment, 53 genes were differentially expressed in roots, of which 15 genes were up-regulated and 35 genes were down-regulated. Only two genes were down-regulated in leaves. Among the up-regulated genes, ZobHLH108 had the highest expression level, which was more than 10 folds of the control. In addition, a total of 27 genes were differentially expressed in both salt and waterlogging stresses, such as ZobHLH128, ZobHLH041, and ZobHLH115 were significantly up-regulated (Fig. 8C).

RT-qPCR

Additionally, 17 DEGs was selected for validation by RT-qPCR. Under salt stress, ZobHLH083, ZobHLH131, ZobHLH043, ZobHLH018, ZobHLH020, and ZobHLH065 were significantly up-regulated in leaves (Fig. 9). ZobHLH142 and ZobHLH003 were up-regulated in both leaves and roots. Under waterlogging stress, the expression levels of ZobHLH032, ZobHLH088, ZobHLH122, ZobHLH126, ZobHLH086, ZobHLH111, ZobHLH108, ZobHLH095, and ZobHLH128 were significantly increased. These results were consistent with the RNA-seq data and highlighted the diverse roles of ZobHLH genes in response to stresses in ginger.

Fig. 9
figure 9

RT-qPCR analysis of 17 differentially expressed genes. Lowercase letters indicate the difference is significant (p < 0.05)

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Subcellular localization of ZobHLHs proteins

In this study, we found that ZobHLH083 and ZobHLH108 exhibited high expression levels in salt stress and waterlogging, respectively, suggesting that these two ZobHLH genes may mediate stress tolerance in ginger. To detect the subcellular localization of ZobHLH083 and ZobHLH108 proteins, fusion proteins with GFP tag were constructed and green florescence was observed. Obviously, the GFP signal of the positive control existed both in the whole cell, whereas ZobHLH083 and ZobHLH108 were mainly localized to the nucleus (Fig. 10). The experimental results were consistent with the predicted results on subcellular localization.

Fig. 10
figure 10

Subcellular localization of ZobHLH083 and ZobHLH108 proteins. ZobHLHs-GFP were transiently expressed in tobacco cells under CaMV 35 S promoter. From left to right, the pictures show GFP signal in dark, bright, and merged field. Scale bar = 20 μm

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Discussion

The bHLH TFs constitute a large family in higher plants, and extensive research has highlighted their crucial roles in plant growth, development, and stress response [42]. Currently, the bHLH family has been identified in multiple species including A.thaliana [43] and banana [44]. In this study, 142 bHLH genes were identified in ginger, which were unevenly distributed across chromosome. The molecular weights of the ZobHLHs proteins identified in ginger ranged from 10615.09 Da to 126549.30 Da, and the isoelectric points ranged from 4.8 to 9.87, which differed from banana, Loropetalum chinense and A. thaliana [44, 45], reflecting evolutionary diversity and adaptation within the bHLH gene family. Protein stability is necessary for life activities and metabolic processes, and unstable proteins may be readily aggregated. While the unstable proteins in complex tend to be stable means that they should interact with other molecules to be stabilized and then perform functions [46]. In this study, all the ZobHLHs are identified to be unstable proteins. Which suggests that ZobHLH in ginger may function as polymers. Predictions of subcellular localization showed that most ZobHLH proteins are located in the nucleus, which is consistent with previous findings in other species [47, 48].

Based on phylogenetic analyses, ZobHLH proteins can be classified into 15 subfamilies. Conserved motifs and gene exon intron structures of ZobHLH proteins in the same subfamily are similar or identical, suggesting a common origin from ancestral bHLH gene. These characteristics in ZobHLH proteins are consistent with previous findings in Arabidopsis, rice and maize [49]. Further analysis showed all the ZobHLHs contain Motif-1 and Motif-2, however, notable differences were observed in conserved motifs between different subfamilies, suggesting evolutionary divergence in both structure and function of ZobHLH proteins. In addition, we identified a diverse range of cis-acting elements in the promoter regions of ZobHLH genes associated with abiotic stress responses. The cis-acting elements are crucial for transcriptional regulation, influencing diverse biological processes and gene expression [50]. Similarly in rye [51] and maize [52], the bHLH promoter likewise contains a variety of cis-regulatory elements related to abiotic stress responses [53].

Gene duplication events are a crucial evolutionary mechanism driving gene expansion in plants [54]. In this study, we identified only one pair (ZobHLH076, ZobHLH077) of tandemly duplicated genes and 42 pairs of segmentally duplicated genes. The presence of segmental duplications on different chromosomes suggests that segmental duplication significantly contributes to the expansion of the ZobHLH gene family. KA/KS analysis of the homologous gene pairs showed that all ZobHLH genes underwent purifying selection, indicating a high degree of evolutionary conservation. Duplicated genes may exhibit functionally redundant, highlighting the importance of functional studies to understand their underlying evolutionary mechanisms [55]. Additionally, constructing interspecies of co-variation maps with two dicotyledonous and four monocotyledonous plants revealed that ginger is closely related to monocotyledonous plants and more distant to dicotyledons plants. Since ginger and banana are tropical monocotyledons that evolved from a common ancestor [23], thus they share the highest number of covariates, suggesting that their genes may have similar structural features and functions.

Proteins function through stable or transient complexes, which are crucial for their biological roles in plants [56]. Exploring protein interactions offers valuable insights into the functions of ZobHLH genes. In this study, a complex network of ZobHLH protein interactions was classified into three tiers based on connectivity. The innermost tier includes the four most interactive proteins (ZobHLH132, ZobHLH103, ZobHLH126 and ZobHLH141), showing their potential pivotal role in the protein interaction network of ginger. In addition, ZobHLH132 was identified as the most highly connected protein, which as a homolog of AtbHLH093 involved in the differentiation of stomatal guard cells [39]. It provides clues to hypothesize that ZobHLH132 performs a similar function in ginger.

The latest research shows that bHLH TFs can be involved in the regulation of vascular bundle development and rhizome expansion in ginger, and it is well known that ginger rhizome size is a major trait determining the ginger yield [57]. In this study, transcriptomic analysis showed that 95 ZobHLH genes were expressed during later stages of rhizome development. Notably, ZobHLH065 and ZobHLH064 were highly expressed at all developmental stages. Phylogenetic analysis showed that ZobHLH065 is closely related to AT1G32640 (MYC2), AT5G46760 (MYC3), AT4G17880 (MYC4), and AT5G46830 (MYC5), all of which contain the bHLH domain and the bHLH-MYC_N domain [58]. These MYC transcription factors, part of the bHLH MYC subfamily, which interact with Jasmonate Zim-domain proteins and are important regulators of JA signaling branches [59], While, JA signaling influences various aspects of plant growth and stress responses, including lateral root development and response to adversity stress in plants [60, 61]. That means the rhizome expansion of ginger may be regulated by ZobHLH065 according to JA signaling pathway. ZobHLH064 is a homolog of AT5G54680 (ILR3) and AT1G51070 (bHLH115), which are involved in metal homeostasis in Arabidopsis growth and development [62]. In addition, eight ZobHLH genes containing regulatory elements for flavonoid biosynthesis were identified. Among which six genes (ZobHLH098, ZobHLH078, ZobHLH022, ZobHLH050, ZobHLH117, ZobHLH126, and ZobHLH069) were expressed during rhizome expansion. These results suggest that ZobHLHs likely play regulatory roles in the ginger development, and especially in the process of rhizome expansion.

In production, ginger belongs to continuous crop, which requires large amounts of chemical fertilizers during cultivation, leading to serious salinization [24], the salt stress can inhibit the growth of ginger seedlings (leaf fresh weight and root fresh weight), which leads to lower yield of ginger [63]. In this study, the expression pattern of ZobHLHs under salt stress and waterlogging treatment showed significant fluctuations. For example, under salt stress, ZobHLH083 was significantly up-regulated in leaves, and phylogenetic analysis showed that this gene was homologous to ICE, which was found to enhance cold tolerance, osmotic and salt tolerance of plants, et al., and play a vitally important transcriptional regulatory role in abiotic stresses [64]. In addition, ZobHLH142 and ZobHLH003 were up-regulated in response to salt stress in both leaves and roots. ZobHLH142 is a homolog of AT4G36930 reported to respond to abiotic stresses in cucumber by regulating cell signaling [65]. ZobHLH003 is a homolog of AT4G16430 involved in balancing plant defense mechanisms against external stresses with [66]. These results suggest that ZobHLHs significantly function in ginger resistance to stresses.

Ginger root system is undeveloped, shallow growth, moisture-loving but intolerance of waterlogging. It is very susceptible to waterlogging, which could seriously affect ginger yield [29]. Waterlogging stress inhibits aerobic respiration and impedes plant growth and development [67]. In this study, a large number of DEGs were identified in the transcriptome of waterlogging stress. For example, ZobHLH108 is homologous to AT2G28160 (FIT1) and significantly induced under waterlogging stress. Studies have shown that FIT1 which is expressed mainly in the root system [68] can integrates iron deficiency and oxidative stress responses, thereby promoting normal plant growth and development [69]. Therefore, we hypothesized that ZobHLH108 perform a similar function under waterlogging. Notably, ZobHLH128 was significantly up-regulated in both salt-stressed leaves and roots under waterlogging treatment compared with the control, highlighting its crucial role in abiotic stress response in ginger. Studies showed that NtbHLH genes are rapidly induced when tobacco subjected to waterlogging stress [70], MfbHLH38 acts positively on plant defense through the ABA pathway and enhances the ROS (reactive oxygen species) scavenging system to reduce oxidative damage [71]. AtbHLH112 responds to abiotic stresses by regulating the expression level of proline dehydrogenase 1 to increase the proline content, and then decrease ROS accumulation and water loss [72]. In conclusion, ZobHLH transcription factors have critical functions in the response to abiotic stresses including salt and waterlogging [73], and ZobHLH083 and ZobHLH108 are potential genes during abiotic stress resistance in ginger. However, research is still needed for further validation.

Conclusion

In this study, 142 ginger ZobHLH genes were comprehensively identified. Bioinformatics analysis revealed their physicochemical properties, gene structures, and expression patterns. The findings indicated that ZobHLH genes play crucial roles in ginger rhizome expansion and in responding to salt and waterlogging stresses, the expression level of ZobHLH065 was highest in the middle and late stages of rhizome expansion, and ZobHLH083 and ZobHLH108 were significantly up-regulated under salt and waterlogging stresses, respectively. These results provided valuable genetic resources and theoretical foundations for molecular breeding and genetic improvement of ginger. The biological functions of these genes and their synergistic effects on enhancing the resistance to environmental stresses in ginger need to be further studied.

Data availability

Ginger rhizome expansion transcriptome data are deposited in the NCBI database (Accession Number: PRJNA788194), and additional data is provided within the manuscript and supplementary information files.

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Funding

This study was financially supported by Open Fund Project of Engineering Research Center of Ecology and Agricultural Use of Wetland, Ministry of Education, Yangtze University (KF202312), Hubei international science and technology cooperation project (2024EHA011), the Hubei Provincial Key R&D Initiative Projects (2022BBA0061, 2021BBA096) and Chongqing Yingcai Excellent Science Project (2022CQYC027).

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Authors and Affiliations

  1. Engineering Research Center of Ecology and Agricultural Use of Wetland, Ministry of Education, and Hubei Key Laboratory of Waterlogging Disaster and Agricultural Use of Wetland, Yangtze University, Hubei, 434025, China

    Deqi Liu, Pang Zhang, Tingting Zhou, Yanbi Wu, Mengping Yuan & Xuemei Zhang

  2. Hubei Key Laboratory of Spice & Horticultural Plant Germplasm Innovation & Utilization, Spice Crops Research Institute, College of Horticulture and Gardening, Yangtze University, Jingzhou, Hubei, 434025, China

    Deqi Liu, Pang Zhang, Tingting Zhou, Yanbi Wu, Mengping Yuan, Xuemei Zhang & Yiqing Liu

  3. College of Smart Agriculture/Institute of Special Plants, Chongqing University of Arts and Sciences, Chongqing, 402160, China

    Yiqing Liu

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Contributions

Y.L. and X.Z. conceived and designed the experiment. D.L. conducted experiments and wrote the manuscript. P.Z. and M.Y. participated in samples collection and experiment conduction. T.Z. and Y.W. performed data analysis. D.L. conducted molecular experiments and X.Z. directed the experiment. X.Z. and Y.L. finalized the manuscript for submission. All authors read and approved the manuscript.

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Liu, D., Zhang, P., Zhou, T. et al. Genome-wide characterization and expression analysis of the bHLH gene family in response to abiotic stresses in Zingiber officinale Roscoe. BMC Genomics 26, 143 (2025). https://doiorg.publicaciones.saludcastillayleon.es/10.1186/s12864-025-11284-8

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