Exploring the Heat Shock Transcription Factor (HSF) Gene Family in Ginger: A Genome-Wide Investigation on Evolution, Expression Profiling, and Response to Developmental and Abiotic Stresses

Ginger is a valuable crop known for its nutritional, seasoning, and health benefits. However, abiotic stresses, such as high temperature and drought, can adversely affect its growth and development. Heat shock transcription factors (HSFs) have been recognized as crucial elements for enhancing heat and drought resistance in plants. Nevertheless, no previous study has investigated the HSF gene family in ginger. In this research, a total of 25 ZoHSF members were identified in the ginger genome, which were unevenly distributed across ten chromosomes. The ZoHSF members were divided into three groups (HSFA, HSFB, and HSFC) based on their gene structure, protein motifs, and phylogenetic relationships with Arabidopsis. Interestingly, we found more collinear gene pairs between ZoHSF and HSF genes from monocots, such as rice, wheat, and banana, than dicots like Arabidopsis thaliana. Additionally, we identified 12 ZoHSF genes that likely arose from duplication events. Promoter analysis revealed that the hormone response elements (MEJA-responsiveness and abscisic acid responsiveness) were dominant among the various cis-elements related to the abiotic stress response in ZoHSF promoters. Expression pattern analysis confirmed differential expression of ZoHSF members across different tissues, with most showing responsiveness to heat and drought stress. This study lays the foundation for further investigations into the functional role of ZoHSFs in regulating abiotic stress responses in ginger.


Introduction
High temperatures have a detrimental effect on plant growth, development, and metabolism, significantly impacting crop yield and quality [1][2][3]. Heat shock transcription factors (HSFs) are crucial proteins involved in regulating plant responses to high temperatures. [4,5]. Previous studies have shown that HSFs possess the ability to enhance plant resistance to high temperatures by engaging with heat shock elements and subsequently activating the expression of downstream genes [6][7][8]. A typical HSF protein contains five conserved oligomerization domains [9]: a DNA-binding domain (DBD), an oligomerization domain (OD) [10,11], a nuclear localization signal domain (NLS), a nuclear export signal domain (NES) [4,12], and a C-terminal activation domain (CTAD) [13,14]. It is worth noting that the CTAD region is only present in certain members of the HSF gene family. However, it plays a crucial role in self-activation by HSFs [15]. Based on the characteristics of their oligomerization structures, the HSF gene family can be divided into three branches: A, B, and C [11].
Ever since the first HSF gene was discovered in yeast, researchers have identified an increasing number of HSFs in various plant species, including Arabidopsis thaliana [16], rice (Oryza sativa) [11], potato (Solanum tuberosum) [17], tomato (Solanum lycopersicum) [18], wheat (Triticum aestivum) [19], and maize (Zea mays) [14]. The respective species possess 21,25,27,24,82, and 25 HSF genes. Overexpression of HSFA9 in tobacco has been shown to enhance the accumulation of carotenoids, chlorophyll, and anthocyanins [20]. It also promotes the expression of genes involved in light morphogenesis (PHYA, PHYB, and HY5), leading to improved photosynthesis and tobacco growth. The modulation of heat shock proteins like HSP70 and HSP90 by HSFs is involved in regulating flowering time and vernalization pathways, thereby suppressing the expression of FLC and promoting flowering [21]. Moreover, the overexpression of the HaHSFA9 gene in tobacco seeds benefits sunflower seeds by enhancing protein accumulation during embryogenesis and promoting callus growth [22].
Additionally, HSF transcription factors are crucial in plants' responses to various abiotic stresses, including heat, oxidative stress, and salt stress. Elevating the levels of HsfA3s in Arabidopsis enhances heat resistance but may increase susceptibility to salt due to changes in proline breakdown pathways [23]. Overexpression of HsfA2 in Arabidopsis leads to the early activation of target genes and improves tolerance to hypoxia [24]. The AtHSFA6b gene is significantly upregulated under high salt and drought conditions, contributing to drought, salt, and heat stress responses mediated by ABA [25]. Moreover, HSFs regulate the expression of other stress response-related genes, influencing plant adaptability. For example, ZmHsf11 negatively regulates oxidative stress-related genes, reducing plant tolerance to heat stress by increasing ROS levels and reducing proline content [26]. HSFA2 enhances the heat tolerance in Arabidopsis by promoting the expression of heat-responsive genes, such as APX2 and HSP [27]. In the case of tomatoes, HsfA3 has been observed to interact with MAP kinases, playing a role in regulating the heat stress response [28]. Additionally, HSFs have been found to participate in somatic embryo maturation and interact with glutathione metabolism [29], ABA signaling [30], and secondary metabolites [31], contributing to enhanced thermotolerance in plants.
Ginger (Zingiber officinale) is a perennial herb belonging to the Zingiberaceae family, renowned for its distinct aroma and strong taste. It serves not only as a valuable medicinal resource due to its high gingerol content but also contains numerous active compounds that possess beneficial properties, including blood pressure regulation, lipid reduction, antioxidation, and immune enhancement [32,33]. Nevertheless, abiotic factors, like high temperature and drought, negatively impact the normal growth and development of ginger, leading to reduced yield and compromised quality [34][35][36]. While HSFs play a crucial role in various abiotic stresses, no reports have documented the presence of HSF gene family members in ginger.
In our previous investigation, we accomplished the successful sequencing and chromosomal assembly of the ginger genome. This achievement has provided a solid foundation for conducting an all-inclusive examination of the HSF gene family in ginger [37]. The current study represents the first identification of 25 members of the HSF gene family from the ginger genome sequence. We analyzed various characteristics of these genes, including gene structures, promoter cis-acting elements, chromosomal locations, conserved motifs, collinearity, replication events, and expression patterns. We further elucidated the evolutionary relationships between ginger and other plant species, such as Arabidopsis thaliana, Triticum aestivum, Oryza sativa, and Musa acuminata. Additionally, we examined the expression profiles of the HSF genes under high temperature stress. Through our bioinformatic analysis, we gained important insights into the ZoHSFs and provided valuable information for further investigations into the role of HSF family genes in ginger's response to abiotic stress.

Genome-Wide Identification of the HSF Family in Ginger
We used the hidden Markov model (HMM) method with the HSF protein domain (PF00447) as the query to identify a total of 25 HSF gene sequences in the Z. officinale reference genome. A variety of gene characteristics have been provided, including protein molecular weights (MW), isoelectric points (pI), and coding sequence (CDS) lengths (Table 1). Within the set of 25 HSF genes, ZoHSF14 is identified as the shortest, encompassing a length of 239 amino acids (aa). Conversely, ZoHSF07 is distinguished as the longest among the group, spanning a length of 585 aa. The MW of the proteins ranged from 27.65 kDa (ZoHSF14) to 73.07 kDa (ZoHSF07), and the pI ranged from 4.62 for ZoHSF17 to 9.62 for ZoHSF02 (Table 1).

Chromosomal Distribution and Classification of the ZoHSF Genes
We found that the 25 identified protein sequences were unevenly located on ten chromosomes of Z. officinale, excluding chromosome 10. To prevent confusion and enhance clarity, we have renamed these sequences from ZoHSF01 to ZoHSF25 based on their specific chromosomal locations. Each chromosome containing the ZoHSF genes varies in the number of genes present. Chromosome 20 had the highest number of ZoHSF genes (five), followed by chromosomes 02 and 08 with four genes each. Whereas chromosomes 2, 18, and 16 each had only one ZoHSF gene ( Figure 1).
In order to examine the categorization of ZoHSF genes, we generated a phylogenetic tree that included A. thaliana with 21 HSF genes and ginger with 25 HSF genes. Utilizing distinctions observed within the HR-A/B domain and phylogenetic connections within the ZoHSF genes, we classified the ZoHSF members into three major groups: HSFA, HSFB, and HSFC ( Figure 2). Out of the three groups, group A consisted of the highest number of ZoHSF members (13), followed by group B (10), with group C exhibiting the fewest members (2). In order to examine the categorization of ZoHSF genes, we generated a phylogenetic tree that included A. thaliana with 21 HSF genes and ginger with 25 HSF genes. Utilizing distinctions observed within the HR-A/B domain and phylogenetic connections within the ZoHSF genes, we classified the ZoHSF members into three major groups: HSFA, HSFB, and HSFC ( Figure 2). Out of the three groups, group A consisted of the highest number of ZoHSF members (13), followed by group B (10), with group C exhibiting the fewest members (2).   In order to examine the categorization of ZoHSF genes, we generated a phylogenetic tree that included A. thaliana with 21 HSF genes and ginger with 25 HSF genes. Utilizing distinctions observed within the HR-A/B domain and phylogenetic connections within the ZoHSF genes, we classified the ZoHSF members into three major groups: HSFA, HSFB, and HSFC ( Figure 2). Out of the three groups, group A consisted of the highest number of ZoHSF members (13), followed by group B (10), with group C exhibiting the fewest members (2).

Gene Structure, Motif Composition
To investigate the ZoHSF gene's structural composition, we conducted a thorough analysis of exon and intron numbers and distribution. With the exception of ZoHSF03, which contains five exons, the remaining 24 members of the ZoHSF genes possess two exons each ( Figure 3C). The ZoHSFs displayed a conservation of the number of exons, although the locations of the exons varied. Similarities were observed in the number and length of exons in both the HSFA and HSFB subgroups, both containing two exons. However, in the HSFC, there was a significant disparity between ZoHSF03, which had more than twice the number of exons as compared to ZoHSF01, indicating a notable difference ( Figure 3C).

Gene Structure, Motif Composition
To investigate the ZoHSF gene's structural composition, we conducted a thorough analysis of exon and intron numbers and distribution. With the exception of ZoHSF03, which contains five exons, the remaining 24 members of the ZoHSF genes possess two exons each ( Figure 3C). The ZoHSFs displayed a conservation of the number of exons, although the locations of the exons varied. Similarities were observed in the number and length of exons in both the HSFA and HSFB subgroups, both containing two exons. However, in the HSFC, there was a significant disparity between ZoHSF03, which had more than twice the number of exons as compared to ZoHSF01, indicating a notable difference ( Figure 3C). The detailed sequence information for each motif can be found in additional file 1 (Table S1 and  Table S2). (C) Exon-intron structures of ZoHSF genes. (D) The cis-acting elements of the ZoHSF promoter region, and different color blocks represent different elements.
To analyze the characteristic regions of the ZoHSF genes, we utilized the online MEME tool to examine the motifs of the ZoHSF genes. Based on the results obtained from MEME, we constructed a schematic diagram representing the structures of the ZoHSF genes and the sequence of 10 motifs. Among the 24 members of the ZoHSF family, motifs  (Table S1 and Table S2). (C) Exon-intron structures of ZoHSF genes. (D) The cis-acting elements of the ZoHSF promoter region, and different color blocks represent different elements.
To analyze the characteristic regions of the ZoHSF genes, we utilized the online MEME tool to examine the motifs of the ZoHSF genes. Based on the results obtained from MEME, we constructed a schematic diagram representing the structures of the ZoHSF genes and the sequence of 10 motifs. Among the 24 members of the ZoHSF family, motifs 1, 2, and 4 were present, while ZoHSF03 only displayed motifs 1 and 4 ( Figure 3B). It is noteworthy that motif 5 was exclusively found in the HSFB, while motifs 6, 7, 8, 9, and 10 were solely detected in HSFA. All members of HSFA possessed motifs 1, 2, 3, 4, and 8 ( Figure 3B). Similarly, the motifs present in HSFB members were consistently comprised of motifs 1, 2, 4, and 5. In terms of the HSFC members (ZoHSF01 and ZoHSF03), they exclusively contained motifs 1, 3, and 4. Generally, each subgroup of ZoHSF members exhibited similar motifs, but there were substantial differences between different subgroups.

Cis-element Analysis of ZoHSFs
To investigate the regulatory mechanisms of ZoHSF genes in abiotic stress responses, we extracted the upstream 2 kb sequences of 25 ZoHSF genes for cis-acting element analysis. Our analysis revealed the presence of multiple cis-acting elements in the ZoHSFs, including environmental response elements and hormone response elements. The diversity of these cis-acting elements suggests that different ZoHSF genes may have distinct potential functions. Most of the promoters of ZoHSF genes contain hormone-responsive elements, such as abscisic acid responsiveness (ABRE), gibberellin responsiveness (GARE-motif, P-box, and TATC-box), salicylic acid responsiveness (TCA-element and SARE), auxin responsiveness (TGA-element, TGA-box, AuxRR-core), and MEJA-responsiveness (CGTCA-motif and TGACG-motif) ( Figure 3D). Of these, MEJA-responsiveness and abscisic acid responsiveness were commonly observed in the promoter region of ZoHSF genes, with 23 ZoHSF genes displaying MEJA responsiveness and 18 ZoHSF genes displaying abscisic acid responsiveness ( Figure 3D). Only two genes (ZoHSF08 and ZoHSF12) contained gibberellin responsiveness ( Figure 3D). Furthermore, the promoter region of ZoHSF genes also exhibited stress responses in the MYB transcription factor binding site (MBS), low temperature responsiveness (LTR), and light responsiveness, suggesting their involvement in different stress responses. Overall, our findings suggest that ZoHSF genes have the ability to participate in various abiotic stress responses and respond to a wide range of hormones.

Evolutionary relationships of the ZoHSFs
To investigate the evolutionary relationships between the ZoHSF genes, an analysis was conducted using MEGA 5.0. The phylogenetic tree was constructed using representative HSF protein sequences from five different species: O. sativa, M. acuminata, Z. officinale, T. aestivum (monocotyledonous plants), and Arabidopsis (dicotyledonous plants). The clustering method based on the HSF of Arabidopsis divided the HSF members into three main groups: HSFA, HSFB, and HSFC ( Figure 4). Group A proteins were found to contain a C-terminal initiation motif (AHA), while the oligomerization motif (HR-A/B) in group B proteins had seven fewer bases as compared to group C proteins. It is interesting to note that each subgroup included HSF family members from all five species, suggesting that the differentiation time of these five species was later than that of HSF transcription factors. As demonstrated in Figure 3B, motif analysis revealed the presence of 10 specific motifs among the HSF members from the five species. Almost all members had motif 1, motif 2, and motif 4. Gene structure analysis indicated that most HSF members had two exons, which is consistent with the findings in ZoHSFs. The results of the evolutionary and motif analyses indicated that HSF from the same subgroup in different species often had similar motif compositions, suggesting potential functional similarities between these proteins.

Genome-Wide Replication Events and Synteny Analysis of ZoHSFs
To investigate the phylogenetic relationship of ZoHSFs in ginger, we compared them with those from four other species: O. sativa, M. acuminata, Arabidopsis, and T. aestivum. The results, shown in Figure 5, indicate collinear relationships between ginger and these species. Gray lines represent all genes with collinear relationships, while red lines represent HSF genes. Among the analyzed species, ginger exhibited syntenic relationships with 17 ZoHSF genes in banana, followed by rice (2), wheat (2), and Arabidopsis (0). The number of collinear gene pairs between ZoHSFs and banana, rice, wheat, and Arabidopsis were identified as 26, 2, 2, and 0, respectively. Notably, two ZoHSF genes (ZoHSF01 and ZoHSF23) in ginger showed collinearity with HSF genes in three species: O. sativa, M. acuminata, and T. aestivum. This suggests that these two ZoHSF genes may have existed prior to the differentiation of these species.

Genome-Wide Replication Events and Synteny Analysis of ZoHSFs
To investigate the phylogenetic relationship of ZoHSFs in ginger, we compared them with those from four other species: O. sativa, M. acuminata, Arabidopsis, and T. aestivum. The results, shown in Figure 5, indicate collinear relationships between ginger and these species. Gray lines represent all genes with collinear relationships, while red lines represent HSF genes. Among the analyzed species, ginger exhibited syntenic relationships with 17 ZoHSF genes in banana, followed by rice (2), wheat (2), and Arabidopsis (0). The number of collinear gene pairs between ZoHSFs and banana, rice, wheat, and Arabidopsis were identified as 26, 2, 2, and 0, respectively. Notably, two ZoHSF genes (ZoHSF01 and ZoHSF23) in ginger showed collinearity with HSF genes in three species: O. sativa, M. acuminata, and T. aestivum. This suggests that these two ZoHSF genes may have existed prior to the differentiation of these species.
To examine the genome-wide replication events of ZoHSF members in ginger, the software McScan X was employed. The analysis results, depicted in Figure 6, indicated a collinear pattern among duplicated gene pairs throughout the ginger genome. The ZoHSF genes was marked with a red line, while the grey line represented all duplicated gene pairs. This analysis revealed that a total of 12 ZoHSF genes were identified as part of six replicated gene pairs. This suggests that these genes may have been generated through genome-wide replication events, highlighting the significant role of such events in the expansion of the ZoHSF gene family. To examine the genome-wide replication events of ZoHSF members in ginger, the software McScan X was employed. The analysis results, depicted in Figure 6, indicated a collinear pattern among duplicated gene pairs throughout the ginger genome. The ZoHSF genes was marked with a red line, while the grey line represented all duplicated gene pairs. This analysis revealed that a total of 12 ZoHSF genes were identified as part of six replicated gene pairs. This suggests that these genes may have been generated through genome-wide replication events, highlighting the significant role of such events in the expansion of the ZoHSF gene family.

Expression Patterns of ZoHSF Genes in Different Plant Tissues and Various Abiotic Stresses
To explore the potential functions of the ZoHSF genes in different developmental stages of ginger organs/tissues and various abiotic stresses, we used RNA-seq data to detect their expression patterns. In different plant tissues, the expression of ZoHSF family members is significantly different ( Figure 7A). Most genes were detected in the all kinds of tissues, such as ZoHSF02, ZoHSF05, and ZoHSF18, while some genes were not detected in these three tissues, such as ZoHSF06 and ZoHSF17. The genes that are not expressed in all tissues may be a pseudogene or have a special temporal or spatial expression pattern that is not detected in our data. Further studies showed that the expression patterns of

Expression Patterns of ZoHSF Genes in Different Plant Tissues and Various Abiotic Stresses
To explore the potential functions of the ZoHSF genes in different developmental stages of ginger organs/tissues and various abiotic stresses, we used RNA-seq data to detect their expression patterns. In different plant tissues, the expression of ZoHSF family members is significantly different ( Figure 7A). Most genes were detected in the all kinds of tissues, such as ZoHSF02, ZoHSF05, and ZoHSF18, while some genes were not detected in these three tissues, such as ZoHSF06 and ZoHSF17. The genes that are not expressed in all tissues may be a pseudogene or have a special temporal or spatial expression pattern that is not detected in our data. Further studies showed that the expression patterns of individual ZoHSF genes were tissue-specific. For example, ZoHSF07, ZoHSF19, and ZoHSF15 were only highly expressed in ginger leaves; ZoHSF21 and ZoHSF12 were only expressed in ginger stems; and ZoHSF25 was only highly expressed in ginger roots. The expression of some genes showed significant trends at different developmental stages. For example, the expression level of ZoHSF18, ZoHSF05, ZoHSF22, and ZoHSF13 decreased first and then increased with flower development, while the expression level of ZoHSF21 and ZoHSF07 increased first and then decreased with flower development ( Figure 7A). To investigate the potential functions of the ZoHSF genes under various abiotic stresses, we used RNA-seq data to detect their expression levels under cold, heat, drought, and salt treatments; ZoHSF14 were not expressed in any of the four treatments. The other 24 ZoHSF genes were induced by at least one stress treatment. Among of them, the expression of 9 genes was induced by cold, that of 11 genes by heat, that of 7 genes by drought, and that of 16 genes by salt ( Figure 7B). Therefore, the greatest number of these genes was induced by salt and the lowest by drought.

Expression Patterns of ZoHSFs under High Temperature and Strong Light Stress
The expression pattern of ginger ZoHSF members under natural high temperature and strong light stress were analyzed using qRT-PCR (Figure 8). Under high temperature and strong light stress, the expression levels of some ZoHSF genes showed a trend of decreasing first and then increasing, including ZoHSF04, ZoHSF06, and ZoHSF07. Some ZoHSF genes can be induced by high temperature and strong light, showing a trend of increasing first and then decreasing, such as ZoHSF05, ZoHSF12, ZoHSF16, and ZoHSF25. The expression levels of ZoHSF05, ZoHSF12, ZoHSF23, and ZoHSF20 reached the highest peak after 3 days of treatment under natural high temperature and a strong light environment, respectively. The expression patterns of these ZoHSF genes under drought treatment were time-specific, and their expression levels peaked after 3 days of treatment and then decreased after 4 days of treatment. To investigate the potential functions of the ZoHSF genes under various abiotic stresses, we used RNA-seq data to detect their expression levels under cold, heat, drought, and salt treatments; ZoHSF14 were not expressed in any of the four treatments. The other 24 ZoHSF genes were induced by at least one stress treatment. Among of them, the expression of 9 genes was induced by cold, that of 11 genes by heat, that of 7 genes by drought, and that of 16 genes by salt ( Figure 7B). Therefore, the greatest number of these genes was induced by salt and the lowest by drought.

Expression Patterns of ZoHSFs under High Temperature and Strong Light Stress
The expression pattern of ginger ZoHSF members under natural high temperature and strong light stress were analyzed using qRT-PCR (Figure 8). Under high temperature and strong light stress, the expression levels of some ZoHSF genes showed a trend of decreasing first and then increasing, including ZoHSF04, ZoHSF06, and ZoHSF07. Some ZoHSF genes can be induced by high temperature and strong light, showing a trend of increasing first and then decreasing, such as ZoHSF05, ZoHSF12, ZoHSF16, and ZoHSF25. The expression levels of ZoHSF05, ZoHSF12, ZoHSF23, and ZoHSF20 reached the highest peak after 3 days of treatment under natural high temperature and a strong light environment, respectively. The expression patterns of these ZoHSF genes under drought treatment were time-specific, and their expression levels peaked after 3 days of treatment and then decreased after 4 days of treatment.

Discussion
Ginger (Zingiber officinale) is widely acknowledged for its economic importan However, it is susceptible to environmental stresses, such as high temperatures, whi can have detrimental effects on its productivity [38]. Previous studies have provided e dence suggesting that heat shock transcription factors (HSFs) play a crucial role in reg lating plant responses to different biotic stresses, including high temperature and droug conditions [39]. While genome-wide analyses of HSF gene families have been conduct in various species with sequenced genomes, there has been no specific investigation in HSF genes in ginger. This study aims to bridge the research gap by identifying the H members of ginger at the genomic level.
The number of HSF gene family members varies among different species. For stance, Arabidopsis thaliana has 21 HSF members [40], goatgrass (Aegilops tauschii) has

Discussion
Ginger (Zingiber officinale) is widely acknowledged for its economic importance. However, it is susceptible to environmental stresses, such as high temperatures, which can have detrimental effects on its productivity [38]. Previous studies have provided evidence suggesting that heat shock transcription factors (HSFs) play a crucial role in regulating plant responses to different biotic stresses, including high temperature and drought conditions [39]. While genome-wide analyses of HSF gene families have been conducted in various species with sequenced genomes, there has been no specific investigation into HSF genes in ginger. This study aims to bridge the research gap by identifying the HSF members of ginger at the genomic level.
The number of HSF gene family members varies among different species. For instance, Arabidopsis thaliana has 21 HSF members [40], goatgrass (Aegilops tauschii) has 19 mem-bers [41], maize (Zea mays) has 25 members [15], and Triticum urartu has 17 members [37]. Previous research has indicated that gene family size can change due to genome recombination and expansion [42]. Throughout the evolution of angiosperms, genome duplications frequently take place, which subsequently contribute to the amplification of gene families [43]. In this study, we identified and classified 25 HSF genes from the ginger genome and designated them as ZoHSFs. They were found to be unevenly distributed over ten chromosomes and classified into three distinct groups (A, B and C) based on phylogenetic analysis. The distribution of HSF gene families in ginger followed a similar pattern to other plant species, with a higher number of HSF genes in groups A and B as compared to group C. This suggests that homologous genes with similar motifs and arrangement may exhibit functional redundancy, while heterologous genes may have similar functions. Furthermore, when comparing the HSF gene family of ginger to Arabidopsis, it was observed that the number of ZoHSFs in groups B and C exceeded that of AtHSF members. This finding suggests that after the early ancestor differentiation of ginger and Arabidopsis, the ZoHSF members in ginger might have undergone replication events.
The expansion of gene families in plant genomes is often attributed to genome replication, tandem replication, and fragment replication, which are considered to be the main drivers of evolution, leading to the emergence of new functions and expression patterns [44]. Previous studies have shown that replication events in certain large gene families, like WRKY, are primarily due to fragment replication and tandem duplication, while gene families, such as MADS and NBS, primarily expand through transposable replication [45]. In the case of ginger, synteny analysis confirmed that the expansion of the ZoHSF gene family primarily resulted from fragment duplication rather than tandem duplication, and a similar expansion occurred in the HSF gene family of Tartary buckwheat [46]. Furthermore, the expression analysis revealed that some duplicated ZoHSF genes exhibited different expression patterns in various tissues and organs. For instance, ZoHSF16 and ZoHSF25 are a pair of duplicated genes, but their expression patterns differ. ZoHSF16 showed high expression levels in mature flowers and the first internode, whereas ZoHSF25 was highly expressed in the root. The specific or redundant cellular functions of these genes during the development of other plants have been observed [47]. Subsequent investigations revealed that the motif positions and compositions of these genes were identical, suggesting that differential gene expression may be attributed to gene mutations that occurred during gene duplication, leading to the loss of certain gene segments [48]. Furthermore, changes in the motif composition during the process of gene replication may also contribute to functional differences [49]. For instance, a comparison of the motif composition between ZoHSF01 and ZoHSF03 highlighted differences, as ZoHSF01 contained motif 2, motif 4, motif 1, and motif 3, whereas ZoHSF03 lacked motif 2 ( Figure 3). This discrepancy in motif composition correlated with divergent expression patterns, with ZoHSF01 showing high expression levels in roots and ZoHSF03 being highly expressed in flowers (Figure 8). To summarize, the variation in gene expression within gene pairs could be attributed to gene mutations or changes in motif composition during gene replication [50].
Monocotyledonous plants include Oryza sativa, Musa acuminata, Zingiber officinale, and Triticum aestivum, whereas Arabidopsis belongs to the dicotyledonous category. Analysis of the phylogenetic tree indicated that most subgroups of ZoHSF contained both monocotyledonous and dicotyledonous representatives (Figure 4). This suggests that HSF genes emerged in both types of plants prior to their differentiation [51]. Additionally, a conserved motif analysis revealed that genes within the same group in ginger shared similar sequence structures. For example, HSFA exclusively possessed motif 8 and motif 9, whereas HSFB contained motif 5; motif 3 was present in both HSFA and HSFB. Moreover, motif 1 and motif 4 were found in all HSF proteins across different groups. This specific arrangement of motifs likely contributes to the distinct functionalities exhibited by each group [52]. Similar findings have been reported in HSF members of other plants, such as Phyllostachys edulis [53], Cucurbita moschata [54], and Camellia sinensis [55].
In plants, introns play a crucial role in evolution and can be gained or lost throughout evolutionary history [56]. Consequently, analyzing gene structures is essential for understanding gene functions. In this study, we examined the gene structure of ZoHSF genes. Figure 3C illustrates that 40% (10/25) of ZoHSF genes lacked introns, while the remaining genes had one to two introns. Interestingly, genes with and without introns were evenly distributed among the three subgroups of the ginger HSF gene family. This phenomenon of intron-less genes has also been observed in other gene families, such as the AP2/ERF gene family and the small GRAS gene family [57]. It is noteworthy that intron-less genes may enable rapid responses to stress and regulation of plant growth and development [58]. The absence of introns in some ginger HSF genes suggests a potential mechanism involving horizontal gene transfer and replication of HSF genes during the evolution originating from nuclear genes [59].
The expressions of metabolic pathway-related genes are regulated by cis-elements in the promoter regions of gene family members [60]. Earlier research has suggested that plant hormones, including abscisic acid (ABA), jasmonic acid (JA), ethylene (Et), and salicylic acid (SA), play a crucial role in modulating the expression of HSFs during abiotic stresses, like high temperature and drought [61][62][63]. During the evaluation of cis-acting elements in the promoter region of ZoHSFs, we observed the presence of diverse elements, such as defense responsiveness elements, low-temperature responsiveness elements, and ABA-responsive elements ( Figure 3D). This indicates that ZoHSF genes not only respond to different stress stimuli but also participate in the regulation of ginger growth and development through the modulation of plant hormone levels [64]. Ginger plants typically have a long flowering cycle, often taking up to 10 years to bloom. The precise mechanism underlying this phenomenon remains unclear. However, our study revealed significant expression of ZoHSF09 in all developmental stages of ginger flowers, including the flower bud, young flower, and mature flower. This finding suggests that ZoHSF09 may be involved in ginger flower development. Another similar gene, ZoHSF02, also shows potential as a candidate gene for regulating ginger flower development. Nevertheless, further experimental verification is necessary to confirm these findings. The rhizome serves as the economic organ of ginger and selecting seed ginger with robust rhizome buds for planting is an effective method to achieve high ginger yields [65]. Based on the expression pattern of ZoHSF01, we observed relatively high expression levels in rhizome buds, the first internode, and the secondary internodes, indicating its potential role in the process of rhizome enlargement. Notably, the expression level of ZoHSF01 was significantly reduced in the third internode of ginger. Thus, further investigations are required to determine whether ZoHSF01 regulates the growth and development of rhizomes.
The similarity of gene function within branches of a multi-species phylogenetic tree has been verified [66]. By analyzing the phylogenetic relationships and collinear analysis of ginger and four other plant HSF members, we can make initial predictions about the function of ZoHSF members in response to abiotic stress. Figure 4 shows that ZoHSF16 and ZoHSF25 are orthologous to AtHSFA1b, as they form a cluster. Previous studies have established that HSFA1b has the ability to enhance the antioxidant defense system of plants by modulating the expression of antioxidant enzymes. This, in turn, mitigates the detrimental effects of oxidative stress on plants [67]. Moreover, HSFA1b also plays a crucial role in regulating the expression of membrane proteins, thereby contributing to the maintenance of cell membrane integrity [68]. Such regulatory mechanisms ensure the stability of both internal and external cell environments, enabling plants to effectively combat drought stress. Consequently, it can be inferred that ZoHSF16 and ZoHSF25 might assist ginger in its resilience against drought stress by controlling the expression of membrane protein, thus preserving cell membrane integrity even under high-temperature conditions. Additionally, qRT-PCR analysis revealed that the expression of ZoHSF16 and ZoHSF25 genes is upregulated in high-temperature and strong-light stress conditions, suggesting their potential involvement in the response to these stressors in ginger. Furthermore, studies have demonstrated that HSFA1d, known for its role in the transcriptional regulation of HSFA2, serves as a critical regulator within the HSF signaling network during environmental stress [69]. Notably, when HSFA1d is overexpressed in cucumber, it can trigger the biosynthesis and signal transmission of endogenous jasmonic acid (JA), resulting in enhanced cold tolerance in cucumber [70]. In our study, we observed that AtHSFA1d and ZoHSF11 clustered together within the same subfamily, implying that they might be orthologous and potentially share similar functions. Interestingly, several stress-related cis-acting elements, including MEJA, ABRE, and LTR, were identified in the promoter of ZoHSF11. Additionally, under high-temperature and strong-light stress conditions, the transcript levels of ZoHSF11 exhibited a substantial increase, indicating its potential role in the stress response of ginger. However, additional experiments are necessary to validate the functions of the aforementioned ZoHSFs.

Plant Materials
In this study, we obtained a locally grown ginger variety called 'Zhugen ginger' from the Chongqing University of Arts and Sciences in Chongqing, China. To simulate the natural conditions in Chongqing, where the highest temperature can exceed 40 • C and the light intensity can reach 103,833 Lux, we exposed two-month-old ginger seedlings to an outdoor environment. The functional leaves of ginger, specifically the third to fifth unfolded leaves from the top to base stem, were collected at 8:30 a.m. and 3:00 p.m. on the first day and at 3:00 p.m. on the second, third, and fourth days. The gathered samples were promptly frozen with the use of liquid nitrogen, followed by preservation at a temperature of −80 • C for subsequent analysis. To ensure accuracy, each sample was prepared with three technical replicates.

Identification and Physicochemical Properties Analysis
The genomic data, including genes, cDNAs, and proteins, of Z. officinale, were obtained from our ginger genome research project [37]. To identify potential candidate genes of the HSF protein in ginger, the HMM data for the HSF protein domain (PF00447) were retrieved from the Pfam website (https://pfam.xfam.org/, accessed on 16 October 2022). A search using the HMM model was conducted on the Z. officinale genome data, and the HSF candidate genes were selected using a threshold value of e < 10 −5 . To validate the identified candidates, the NCBI BatchCDD search was employed. The physical and chemical properties of the newly identified ZoHSF members were analyzed using ProtParam (https://web.expasy.org/protparam/, accessed on 16 October 2022). The positions of the ZoHSF members were then mapped onto the reference genome and named according to their chromosome positions using TBtools [71].

Phylogenetic Analysis
To align the full-length protein sequences of reported HSF in A. thaliana and the newly identified ZoHSFs, MAFFT was utilized with the default parameters [72]. The alignment results were then used to construct a neighbor-joining (NJ) tree using MEGA X. The parameters for constructing the NJ tree were set as follows: Poisson model, pairwise deletion, and 1000 replicates for bootstrap values [73]. Additionally, a multi-species phylogenetic evolutionary tree was built, incorporating protein sequences of ZoHSF, A. thaliana, T. aestivum, O. sativa, and M. acuminata HSFs obtained from the UniProt database (https://www.uniprot.org/, accessed on 16 October 2022).

Genetic Structure, Motifs Composition, Gene Duplication and Cis-Acting Elements
The exon-intron structure of the ZoHSF genes was determined using the Gene Structure Display Server (GSDS: http://gsds.cbi.pku.edu.cn/, accessed on 17 October 2022). To identify conserved motifs within the ZoHSF proteins, we employed the MEME online tool available at http:/meme.nbcr.net/meme/intro.html (accessed on 17 October 2022). Furthermore, the plantCARE software (https://bioinformatics.psb.ugent.be/webtools/plantcare/ html/, accessed on 17 October 2022) was utilized to predict the cis-acting elements within the promoters of ZoHSFs. An analysis of gene duplication events in ginger HSF members was conducted using the multiple collinear scanning toolkits (MCScanX), while the Dual Synteny software revealed the homologous relationship between the ginger HSF gene and those of A. thaliana, T. aestivum, O. sativa, and M. acuminata.

Gene Expression and qRT-PCR Analysis
In a previous study conducted by our research group, we conducted transcriptome analysis of ginger in various tissues subjected to abiotic stresses, including cold, heat, high temperature, and drought [44]. Among the samples collected, various tissues of ginger were included, such as 6-month-old flowers, buds, pedicels, stems, rhizome buds, and rhizomes. Additionally, the first, second, and third internodes, functional leaves (the third leaf from the top to the base of the stem), leaf buds, and roots were also included. For the drought and salinity treatments, the plants were irrigated with a solution containing 15% PEG6000 to induce drought stress and 200 mM NaCl for salinity stress. Heat stress treatment involved subjecting the ginger plantlets to 40 • C, while cold stress treatment involved exposing them to 4 • C. Following these treatments, leaf samples were collected at specific time points, including 0, 1, 3, 6, 12, 24, and 48 h after cold, drought, and salt treatments. Additionally, leaves were collected at corresponding time intervals of 0, 1, 3, 6, 12, and 24 h following the heat treatment. This analysis allowed us to investigate the expression patterns of ZoHSF members in different tissues and under abiotic stress conditions. To visualize these expression patterns, we utilized the HeatMap program of the TBtools software. The TRIzol kit (Invitrogen, California, USA) was used for the extraction of total RNA from all samples, and cDNA was synthesized from RNA using the Evo M-MLV reverse transcription kit (Accurate Biology, Shanghai, China). Primers for ZoHSF members (additional file 2: Table S3) were designed using the Primer premier 5.0 software. The selected ZoHSF genes were subjected to qRT-PCR to analyze their response to abiotic stress. The ZoTUB2 (ZOFF_005593) gene served as an internal control, and each qRT-PCR experiment was performed in triplicate using the CFX96 Real-Time System (Bio-Rad). The following protocol was followed: an initial denaturation step at 95 • C for 30 s, followed by a series of 40 cycles, each consisting of denaturation at 95 • C for 10 s and annealing at 60 • C for 30 s. To ensure biological accuracy, each reaction was performed three times as a replicate. The expression data were quantified using the 2 −(∆∆Ct) method [74].

Conclusions
In conclusion, we identified a total of 25 members of the ZoHSF gene family in the ginger genome, and they exhibited uneven distribution across ten chromosomes. These ZoHSF members were classified into three groups, namely HSFA, HSFB, and HSFC. Additionally, our analysis revealed that 12 ZoHSF genes likely originated from duplication events. Through promoter analysis, it was observed that hormone response elements, such as MEJA responsiveness and abscisic acid responsiveness, were prevalent among the cis-elements associated with abiotic stress response in ZoHSF promoters. Furthermore, by examining the phylogenetic and collinear relationships between ginger and other plant HSF members, we gained preliminary insights into the function of various ZoHSF genes. Specifically, our findings showed that ZoHSF16 and ZoHSF25 are orthologous to AtHSFA1b and likely play a role in ginger's response to high temperature and strong light stress. The duplicated gene pairs ZoHSF16 and ZoHSF25 suggest functional similarities between them. Moreover, our results suggest that ZoHSF11, which shares orthology with AtHSFA1d, may be involved in ginger's stress response process. This is supported by its increased expression under high-temperature and strong-light stress conditions, as well as the presence of stress-related cis-acting elements on its promoter. Overall, this research enhances our understanding of the molecular mechanisms underlying stress responses in ginger plants and paves the way for further investigation of HSF genes in this species.
Supplementary Materials: The following supporting information can be downloaded at https: //www.mdpi.com/article/10.3390/plants12162999/s1, additional file 1: Table S1: Analysis and distribution of conserved motifs in other four plants HSF proteins. Table S2: Analysis and distribution of conserved motifs in Zingiber officinale HSF proteins; additional file 2: Table S3: The primer sequences for qRT-PCR used in this study.

Data Availability Statement:
The data utilized in this study are currently not publicly available as they are part of the ginger genome and have not been released. However, the data presented in this study can be made available upon request from the corresponding author.