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Genome-wide identification, phylogeny and expression analysis of Hsf gene family in Verbena bonariensis under low-temperature stress

BMC Genomics volume 25, Article number: 729 (2024) Cite this article

Abstract

Background

The heat shock transcription factor (Hsf) is a crucial regulator of plant stress resistance, playing a key role in plant stress response, growth, and development regulation.

Results

In this study, we utilized bioinformatics tools to screen 25 VbHsf members, which were named VbHsf1-VbHsf25. We used bioinformatics methods to analyze the sequence structure, physicochemical properties, conserved motifs, phylogenetic evolution, chromosome localization, promoter cis-acting elements, collinearity, and gene expression of Hsf heat shock transcription factor family members under low-temperature stress. The results revealed that the majority of the Hsf genes contained motif1, motif2, and motif3, signifying that these three motifs were highly conserved in the Hsf protein sequence of Verbena bonariensis. Although there were some variations in motif deletion among the members, the domain remained highly conserved. The theoretical isoelectric point ranged from 4.17 to 9.71, with 21 members being unstable proteins and the remainder being stable proteins. Subcellular localization predictions indicated that all members were located in the nucleus. Phylogenetic analysis of the Hsf gene family in V. bonariensis and Arabidopsis thaliana revealed that the Hsf gene family of V. bonariensis could be categorized into three groups, with group A comprising 17 members and group C having at least two members. Among the 25 Hsf members, there were 1–3 exons located on seven chromosome fragments, which were unevenly distributed. Collinearity analysis demonstrated the presence of seven pairs of homologous genes in the VbHsf gene family. The Ka/Ks ratios were less than one, indicating that the VbHsf gene underwent purification selection pressure. Additionally, nine genes in V. bonariensis were found to have collinearity with A. thaliana. Promoter analysis revealed that the promoters of all VbHsf genes contained various types of cis-acting elements related to hormones and stress. Based on RNA-seq data, qRT-PCR analysis of six highly expressed genes was performed, and it was found that VbHsf5, VbHsf14, VbHsf17, VbHsf18, VbHsf20 and VbHsf21 genes were highly expressed at 12 h of low-temperature treatment, and the expression decreased after 24 h, among which VbHsf14 was up-regulated at 12 h of low-temperature by 70-fold.

Conclusions

Our study may help reveal the important roles of Hsf in plant development and show insight for the further molecular breeding of V. bonariensis.

Peer Review reports

Background

Heat shock transcription factor (Hsf) is an important regulator of plant stress translation, which regulates gene expression by binding to heat shock response elements (HSE) in the promoter regions of target genes, and then controls the transcription and translation process of heat shock proteins, and responds to various biotic and abiotic stresses [1]. Moreover, Hsf also plays an important role in plant growth and development [2, 3]. Studies have demonstrated that the typical Hsf in eukaryotes consists of five conserved domains. These domains include the DNA-binding domain (DBD), the oligomerization domain (OD), the C-terminal activation domain (CTAD), the nuclear localization signals (NLS), and the nuclear export signals (NES). Among these domains, the DBD and OD are the most conserved and are responsible for accurately identifying and binding to the target genes’ promoters, respectively. The NLS and NES domains ensure the transport of Hsf into the nucleus and maintain the appropriate distribution of genes between the nucleus and cytoplasm. The CTAD contains a transcriptional activation function and directly regulates the expression of relevant genes during heat shock. The transcriptional activation function is mediated by a short activating peptide motif known as the AHA motif within the CTAD [4,5,6,7] .

V. bonariensis belongs to the Verbenaceae family and the genus Verbena [8]. It is commonly used as a background plant in flower borders and is widely employed in landscape construction. In the karst region of Guizhou, V. bonariensis demonstrates adaptability to karst soil and plays a crucial role in many scenic spots, park green spaces, and urban greening projects [9]. Currently, it is cultivated in numerous scenic spots such as Huaxi Gaopo Township (Genting Flower Sea), Wild Jade Sea View Area (Pheasant Ping starry sky flowers), Dushan Laran Town, Tongzi Loushanguan,, Xingren Fangmaping, and more. Especially in recent years, Guizhou Province has made significant efforts in developing the ecological industry and implementing province-wide ecological construction and protection systems. This includes the establishment of urban green spaces (with a green coverage rate of 35% in urban built-up areas), wetland parks, forest parks, mountain parks, etc. With its exceptional qualities as a garden ornamental plant, V. bonariensis holds immense potential for widespread application.

Low temperatures are a significant factor that hinders plant growth and distribution [10]. The Guizhou karst area specifically faces the challenge of continuous low temperatures and rainy weather below 4℃ in winter, as well as cold problems in late spring. Unfortunately, V. bonariensis is not tolerant to low temperatures and exhibits slow growth below 10℃ [11]. It cannot naturally survive winters below 5℃ [11]. To overcome this issue in production, conventional methods such as greenhouse cultivation and the application of plant hormones are often employed to facilitate overwintering. However, these methods require substantial manpower and material resources, increasing the costs associated with raising, managing, and maintaining V. bonariensis seedlings. Consequently, the low temperature resistance of V. bonariensis has become a bottleneck in its further promotion and application in Guizhou. Therefore, it is crucial to preserve a sufficient reserve of genes related to cold resistance, specifically isolating and functionally analyzing multi-valent genes and transcription factors with a ‘master switch’ function for plant cold resistance breeding [12].

As a tropical crop, V. bonariensis has the characteristics of thermophilic but not cold-tolerant, but the research on Hsf gene of V. bonariensis has not been reported. In this study, the whole genome identification of the V. bonariensis Hsf gene family was carried out, and the sequence characteristics, chromosome localization and genome replication events of the gene family members were analyzed by bioinformatics methods. Combined with the transcriptome data of the leaves of V. bonariensis under cold stress, the expression pattern of VbHsf gene under cold stress was analyzed, which laid a foundation for further study of the cold response mechanism and biological function of V. bonariensis Hsf gene family members, and provided a theoretical basis for further study of the role of this family in the regulation of V. bonariensis response to stress.

Results

Identification and physicochemical analysis of Hsf gene family members in V. bonariensis

Through identification and screening, a total of 25 Hsf gene family members were identified in V. bonariensis. These members were designated as VbHsf1-VbHsf25 based on their distribution on chromosomes (Table S1). The amino acid lengths of the VbHsf proteins ranged from 110 to 474 aa, with molecular weights ranging from 12,410.88 to 52,487.92. The theoretical isoelectric points of these proteins ranged from 4.17 to 9.71. Among the 25 members, 18 were classified as acidic proteins and seven were classified as basic proteins. The analysis of protein hydrophilicity and hydrophobicity revealed that the Hsf protein with the weakest hydrophilicity had a value of -0.968, while the Hsf protein with the strongest hydrophilicity had a value of -0.119; both of these proteins were classified as hydrophilic. The instability coefficient indicated that most of the Hsf gene family members were unstable proteins (instability coefficient > 40). However, four members, namely VbHsf8, VbHsf13, VbHsf19, and VbHsf22, were found to be stable proteins (instability coefficient < 40), with VbHsf19 having the lowest instability coefficient of 29.60. The fat coefficient ranged from 52.18 to 78.33. Analysis of transmembrane domains showed that only VbHsf19 possessed a single transmembrane domain, while the remaining 24 members did not have any transmembrane domains. The subcellular localization of the VbHsf proteins was determined to be in the nucleus. Secondary structure prediction revealed that the Hsf family mainly consisted of α-helix and random coil structures, comprising more than 67% of the protein’s amino acid composition. In contrast, the proportion of extended chain structures (β-sheet) and β-turn structures was relatively low (Table S2). These findings suggest that α-helix and random coil structures are prevalent in Hsf proteins, while extended chain and β-turn structures are sparsely distributed throughout the protein.

Analysis of domain composition and conserved motifs of Hsf family proteins in V. bonariensis

To investigate the structural characteristics of VbHsf genes, we analyzed the exon, 5’ UTR, and 3’ UTR sequences of 25 VbHsf genes. The analysis aimed to explore the genetic structural features of these genes. The findings revealed that VbHsf genes generally comprised 1–3 exons. Specifically, VbHsf6 and VbHsf13 contained a single exon, VbHsf15 had three exons, and the remaining VbHsf genes had two exons. Among the VbHsf genes, only VbHsf1 and VbHsf10 had a 3’ UTR sequence, while seven VbHsf genes (VbHsf7, VbHsf16, VbHsf18, VbHsf20, VbHsf23, VbHsf24, and VbHsf25) possessed both 5’ UTR and 3’ UTR sequences. Notably, the remaining 16 VbHsf genes lacked both 5’ UTR and 3’ UTR sequences. Furthermore, closely related genes exhibited similar exon-intron structures (Fig. 1).

We utilized MEME to conduct a further analysis of conserved motifs in members of the Hsf protein family. The findings revealed that the motif types and arrangement in all VbHsf genes were largely similar, but some motifs exhibited varying degrees of deletion. Notably, VbHsf8 contained only two conserved motifs, indicating the most severe deletion. The DNA binding domain (DBD) and oligomerization domain (OD) were identified as the most conserved domains. To investigate the sequence conservation characteristics among members of the Hsf gene family, the amino acid sequences of the 25 Hsf genes obtained in this study underwent multiple sequence alignment. The alignment demonstrated that all amino acid sequences included a DBD domain composed of motif 3, motif 1, and motif 2. This DBD domain, located at the N-terminus of the VbHsf gene, spans approximately 100 amino acids and is characterized by three α-helices (α1-α3) and four β-sheets (β1-β4). These structural features enable specific recognition and accurate localization of heat stress elements. Similar observations have been made in other plant species. Besides DBD, the HR-A/B region, which exhibits a predicted coil-like structure, is crucial for Hsf-Hsf interaction and trimer formation [13]. Most VbHsf genes also possess HR-A/B. Moreover, compared to class B and class C Hsfs, class A Hsfs have a longer HR-A/B region (Fig. 2, Table S3).

The Hsf protein family also includes other domains such as NLS, NES, and AHA. NLS and NES play crucial roles in the intercellular distribution and interaction of Hsf genes within the nucleus and cytoplasm [14]. For NLS prediction, we utilized the cNLS Mapper software, while the LocNES software was employed for NES prediction. Most VbHsfs were found to contain NES, whereas NLS was absent in class C genes, certain class B genes, and some class A genes. Specifically, 13 (52%) VbHsfs contained the NLS domain, while 22 (88%) VbHsfs contained the NES domain. The AHA motif is unique to most class A genes. In particular, the AHA motif (motif 10) is exclusive to class A VbHsf proteins, and among them, VbHsf2, VbHsf3, VbHsf9, and VbHsf25 proteins possess the AHA motifs (Table S3).

Fig. 1
figure 1

Evolutionary relationship, gene structure and distribution of conserved motifs of Hsf gene family members in V. bonariensis

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Fig. 2
figure 2

Multiple sequence alignment of DNA binding domains and the HR-A/B regions (OD) of VbHsf

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Phylogenetic tree and chromosome mapping of VbHsf gene family in V. bonariensis

In order to investigate the homologous evolutionary relationship between V. bonariensis and other species, we utilized the Hsf gene family of A. thaliana as a comparative reference. The identified Hsf gene family of V. bonariensis (25) and the Hsf gene family of A. thaliana (21) were then constructed using the maximum likelihood method to create a phylogenetic tree (Fig. 3). The results revealed that the VbHsf family genes were categorized into three distinct groups: group A, group B, and group C. Group A consisted of 17 VbHsf genes and 15 AtHsf genes, group B contained six VbHsf genes and five AtHsf genes, and the least number of genes was found in group C, which included two VbHsf genes and one AtHsf gene. Notably, the number of Hsf genes in groups A, B, and C were similar between the two species, whereas the number of Hsf genes in the three groups differed significantly between V. bonariensis and A. thaliana.

The TBtools tool was employed to create a gene chromosome distribution map based on the chromosome location information of the gene family members. The analysis results (Fig. 4) demonstrated that all VbHsf genes were unevenly distributed across 7 chromosome segments. The number of genes on each chromosome ranged from 1 to 6. Chr3 exhibited the highest gene count (6), followed by Chr1 (5). Chr2 and 5 each contained four genes, while Chr4 contained 3 genes. Chr7 contained 2 genes and Chr6 contained 1 gene. Holub defined a chromosomal region with more than two genes within a 200 kb range as tandem repeats [15]. In our study, we identified a pair of tandem repeat genes in VbHsf, which are highlighted in blue.

Fig. 3
figure 3

Phylogenetic tree of Hsf gene family members between V. bonariensis and A. thaliana

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Fig. 4
figure 4

Chromosomal location of VbHsf gene family members in V. bonariensis

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Analysis of cis-acting elements in the promoter of hsf gene family in V. bonariensis

To investigate the potential function of the VbHsf gene in V. bonariensis, the Plant CARE tool was utilized to predict various cis-acting elements within a 2000 bp region upstream of the gene start site. The presence of diverse cis-acting elements suggests that different genes may have a range of potential functions. Interestingly, the VbHsf family exhibited a variety of cis-acting elements in response to environmental and hormone signals (Fig. 5), indicating a complex expression regulation mechanism. For instance, the cis-acting element LTR, known to be involved in low temperature response [16], was identified in VbHsf1, VbHsf10, VbHsf11, VbHsf12, VbHsf13, VbHsf15, VbHsf18, VbHsf19, VbHsf21, VbHsf22, and VbHsf25. Furthermore, eight VbHsf genes contained defense and stress response elements (TC-rich repeats). Additionally, 23 VbHsf genes possessed drought, high temperature, and low temperature response elements (MYB). The anaerobic sensing element (ARE) was found in 18 VbHsf genes, while the WUN-motif and AAGAA-motif (stress response elements) were identified in 17 VbHsf genes. Moreover, 24 VbHsf genes contained cis-acting elements (MYC) related to abscisic acid resistance, drought resistance, freezing resistance, and cold resistance. The cis-acting elements (MBS) associated with drought induction were present in 13 VbHsf genes, while the W-box (biotic stress response element) was found in 12 VbHsf genes. Jasmonic acid response elements (CGTCA-motif/TGACG-motif) were present in 19 VbHsf genes, and abscisic acid signal response elements (ABRE) were identified in 22 VbHsf genes. Furthermore, ethylene response elements (ERE) and salicylic acid response elements (TCA-element) were found in 19 and 10 VbHsf genes, respectively. The auxin response element (TGA-element) was identified in seven VbHsf genes. Additionally, there were nine, six, and foru VbHsf genes containing cis-acting elements TATC-box, P-box, and GARE-motif, respectively. It is worth noting that the promoters of all VbHsf genes contain various types of cis-acting elements related to hormones and stress. Among these elements, ABRE and stress response elements MYB and MYC were the most abundant, suggesting that VbHsfs may respond to plant hormones and stress, such as salt, drought, low temperature stress, and ABA induction. However, gene expression is regulated by a diverse array of elements, indicating complex regulatory mechanisms that contribute to the diversification of gene functions. Further verification is required to determine the specificity of gene functions.

Fig. 5
figure 5

Cis-acting element of V. bonariensis Hsf gene family

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Colinearity analysis of hsf gene family members in V. bonariensis

To investigate the conservation of the Hsf gene family members in the evolutionary process of V. bonariensis, a collinearity analysis was conducted on the Hsf gene family members (Fig. 6). The analysis revealed the presence of seven pairs of highly homologous gene pairs within the Hsf gene family of V. bonariensis. Subsequently, the values of Ka (non-synonymous mutation rate) and Ks (synonymous mutation rate) were calculated for these 7 pairs of highly homologous gene pairs (Table S4). The Ka/Ks ratio is commonly used to determine whether a gene encoding a protein is subject to selective pressure. A Ka/Ks ratio > 1 generally indicates a positive selection effect, while a Ka/Ks ratio = 1 suggests neutral selection, and a Ka/Ks ratio < 1 implies purifying selection. In this study, the Ka/Ks values for all seven pairs of homologous gene pairs in the table were found to be less than one, indicating that these gene pairs evolved under the pressure of purifying selection in V. bonariensis.

To further investigate the origins, evolutionary history, and potential functions of the Hsf gene in V. bonariensis, we conducted a comparison of the genomic sequences between V. bonariensis and A. thaliana. We identified a total of 15 pairs of collinear relationships between the two species, with 9 genes from V. bonariensis and 11 genes from A. thaliana (Fig. 7). Notably, we observed that four genes (AtHsfA6a-VbHsf1/VbHsf24, AtHsfA6b-VbHsf1/VbHsf24, AtHsfA7b-VbHsf1/VbHsf24, and AtHsfB3-VbHsf13/VbHsf23) in A. thaliana exhibited multiple pairings with V. bonariensis genes. Similarly, three genes (VbHsf9-AtHsfA1a/AtHsfA1d, VbHsf2-AtHsfA1b/AtHsfA1e, VbHsf1-AtHsfA6a/AtHsfA6b/AtHsfA7b, and VbHsf24-AtHsfA6a/AtHsfA6b/AtHsfA7b) in V. bonariensis were paired with two or three genes in A. thaliana. It is important to note that all these genes belong to the same subfamily, suggesting a shared ancestral origin before subsequent evolution.

Fig. 6
figure 6

Colinearity relationship of Hsf gene family members in V. bonariensis

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Fig. 7
figure 7

Homology analysis of Hsf gene between V. bonariensis and A. thaliana

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Differences in the expression of hsf gene family members in V. bonariensis

In order to further investigate the regulatory role of Hsf gene family members in the growth, development, and response to low temperature stress in V. bonariensis, this study analyzed and visualized the transcriptome data of four different parts and low temperature stress conditions from the V. bonariensis database (Figs. 8 and 9). The results revealed distinct expression patterns of VbHsf in various tissues. Specifically, three VbHsf genes (VbHsf6, VbHsf5, and VbHsf21) exhibited high expression levels in almost all tissues, indicating their potential importance in multiple processes. Additionally, all VbHsf genes, with the exception of VbHsf8, were found to be expressed in at least one of the analyzed tissues. Notably, the significant upregulation of VbHsf23 in flowers compared to other tissues suggests its potential role in flower development and function.

The expression patterns of the Hsf gene family in V. bonariensis under low temperature stress can be categorized into three groups. Among these, 13 members showed an increase in expression levels under cold stress, with seven members being significantly up-regulated. On the other hand, six members exhibited a decrease in expression levels, while the remaining 6 members did not respond to cold stress. The up-regulated expression levels of seven VbHsf members were found to contain homeopathic elements associated with abiotic stress (LTR, MYB, MYC, WUN-motif), suggesting their potential involvement in the response to cold stress and cold resistance. The distribution of significantly differentially expressed VbHsf genes indicated that class A and class C VbHsf genes were prominently up-regulated, highlighting their crucial regulatory role in response to low temperature stress. Conversely, the expression of class B VbHsf genes was more inhibited under low temperature stress. These findings provide a theoretical foundation for further investigations into the mechanism of low temperature stress response in V. bonariensis.

Fig. 8
figure 8

Expression difference of Hsf gene family members in different parts of V. bonariensis

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Fig. 9
figure 9

The expression changes of V. bonariensis after low temperature treatment

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Examination of the expression of VbHsf genes by qPCR under low-temperature stress

In order to verify the RNA-seq results of V. bonariensis under low-temperature stress, we performed qRT-PCR analysis of six highly expressed genes based on RNA-seq data (Fig. 10). It was found that the expression profiles shown by qRT-PCR were similar to those obtained by RNA-seq (Fig. 9). The VbHsf5, VbHsf14, VbHsf17, VbHsf18, VbHsf20 and VbHsf21 genes showed high expression at 12 h of low temperature treatment, and then the relative expression decreased after 24 h, which showed an overall tendency of increasing and then decreasing; the relative expression of VbHsf14 and VbHsf18 was significantly increased at 6 h of treatment; the VbHsf14 gene had relatively high expression after 3, 6, 9, 12 and 24 h of treatment, which was the highest among all genes. This suggests that these genes may be the key regulators of low- temperature stress response in V. bonariensis.

Fig. 10
figure 10

Expression analysis of six VbHsf genes in V. bonariensis leaves under cold stress. Lowercase letter(s) above the bars indicate significant differences (α = 0.05, LSD) among the treatment

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Discussion

Hsf transcription factors are widely distributed in plants and play crucial roles in plant growth, development, and stress response [17]. Extensive studies have been conducted to identify and analyze the members of the Hsf family in various plant species. Genome-wide investigations have revealed that A. thaliana, Vitis vinifera (grapes), Beta vulgaris (sugar beets), and Populus (poplars harbor) 21, 19, 13, and 28 members of the Hsf transcription factor gene family, respectively [18], at the same time, these studies have also shown that the same gene family has some differences in the number of gene family members, gene structure and gene function in different species. However, the Hsf gene has not been identified in the whole genome of V.bonariensis, and its function has not been reported. Therefore, the launch of the analysis of the VbHsf gene family of V. bonariensis is conducive to in-depth study of the unique biological functions of the VbHsf gene.

The majority of studies have consistently reported that members of the Hsf gene family are predominantly hydrophilic proteins and exhibit instability. For example, 17 members of the Hsf gene family identified in carnation, were all hydrophilic and mostly unstable proteins. All 25 Hsf identified in V. bonariensis in this study were also hydrophilic and mostly unstable proteins [19]. The theoretical isoelectric points of the Hsf gene family members in most studies were very similar, ranging from 4.65 to 9.36, in the present study, the theoretical isoelectric point of VbHsf was found to be 4.17–9.71, which is similar to the results of previous studies [20, 21]. Structural differences among gene family members have been identified as valuable for elucidating functional differentiations across species. For instance, Renjun Qu et al. [22] identified Motifs 1, 2, and 3 in the Hsf gene family of Salvia miltiorrhiza, excluding SmHsf11. Similarly, Lulu Wang et al. [23] discovered that Motifs 1, 2, and 3 were the most widely distributed and conserved in the Hsf protein sequence of Passiflora edulis Sims (passion fruit). In this study, all VbHsfs subpopulations were found to contain motifs 1, 2, and 3 encoding the DBD domain, which displayed a highly conserved triple helix bundle structure. This indicating a high degree of conservation in the Hsf gene family during evolution with notable differences. Additionally, this study identified other conserved motifs unique to the members of the Hsf gene family in V. bonariensis, suggesting the potential importance of these motifs in functional differentiation within this species. The specificity of these structural elements may contribute to the specificity of functions observed in different groups. Furthermore, previous studies have revealed the presence of 2–5 exons in the Hsf gene family of S. miltiorrhiza [22] and 1–3 exons in the Hsf gene family of maize [21], which aligns with the findings of this study regarding the exonic composition of the Hsf gene family in V. bonariensis.

The phylogenetic tree is a useful tool for elucidating the evolutionary relationships among different species or genes. For instance, Kai Zhao et al. [24] categorized the phylogenetic tree of poplar into group A, group B, and group C based on the grouping in A. thaliana. Likewise, Qi Wang et al. [25] divided the Hsf gene family in Arachis hypogaea (peanut) and A. thaliana into three groups (A, B, and C) and 14 subgroups (A9-A1, B4-B1, and C1) in the phylogenetic tree. In this study, the phylogenetic trees of Hsf members from V. bonariensis and A. thaliana were also clustered into three categories and 14 subgroups, mirroring the grouping observed in A. thaliana Hsf gene family members [26]. This suggests that homologous genes with similar motif types and arrangement sequences may exhibit functional redundancy, while heterologous genes may share similar functions. Furthermore, the varying number of genes in each group indicates an uneven distribution of Hsf gene family members, suggesting a wide range of diversity among them. These genetic differences and diversity likely contribute to the diverse functions of Hsf genes, providing valuable resources for studying the Hsf gene in V. bonariensis.

Replication within gene family members plays a significant role in plant evolution, and collinearity can be used to predict homologous gene sequences during evolutionary processes. In Cucumis sativus (cucumber), it was observed that 96% of duplicated gene pairs exhibited Ka/Ks values below 1, indicating strong purification selection [27]. Similarly, in sunflower, the Ka/Ks ratios of Hsf gene family members were also lower than one [28]. In this study, it was observed that seven pairs of homologous Hsf genes in V. bonariensis had Ka/Ks values below one, suggesting that these gene pairs underwent purification selection, which may have contributed to the conservation of the Hsf gene structure throughout evolution. Additionally, a collinearity relationship was identified between nine VbHsfs and 11 AtHsfs, showing some homology, suggesting that they may have evolved from the same ancestor, providing insights into the potential functions of VbHsf genes.

Previous studies have emphasized the crucial role of Hsf in the response to biotic and abiotic stress, but the identification of cold-related Hsf genes has been limited [29]. For instance, Jing Bin et al. [30] demonstrated that certain OfHsf members were induced by cold stress in Osmanthus fragrans. In A. thaliana, Park et al. [31]. identified 30 transcription factors (TFs) that were rapidly induced by low temperatures, referred to as “the first wave of cold-induced TFs,” which included AtHsfC1. Furthermore, studies have revealed that the promoters of Hsf gene family encompass cis-acting elements associated with stress. Qi Zhang et al. [32] found that 20% (six out of 30) of the Hsf gene families contained stress-related cis-acting elements such as LTR, ARE, and MBS. Pan-Song Li et al. [5] discovered that each soybean Hsf member’s promoter carried one or more MYB and MYC cis-acting elements. In our investigation, it was observed that the promoters of all VbHsf genes contained various stress-related cis-acting elements, including LTR, MYB, MYC, WUN-motif, among others. This suggests that V. bonariensis potentially regulates plant abiotic stress adaptability. Changwei Shen et al. [33] determined that 44% of CmHsfs (16 genes) were significantly up-regulated in pumpkin under cold treatment. Leveraging the genome and transcriptome data of V. bonariensis, we analyzed VbHsf and found that the expression of VbHsf (seven genes) was significantly up-regulated by 28% under low temperature stress.

Plants can respond to abiotic stress and regulate growth and development through tissue-specific gene expression [34, 35]. Liu et al. [36] and Song et al. [37] found that Hsf genes were differentially expressed in Medicago sativa L., Chinese cabbage, respectively. In V. bonariensis, we found that VbHsf showed specific expression patterns in different tissues, for example, VbHsf23 was more highly expressed in flowers, suggesting that this gene may play an important role in the growth and development of flowers. Hsf has been shown to have an important role in plant response to abiotic stresses [38,39,40]. Li et al. [41] found in Dianthus caryophyllus that most of the dcahsf were responsive to heat stress, while some genes were down-regulated under cold stress. In Cymbidium ensifolium, the expression of CeHSF7, CeHSF11, CeHSF14, CeHSF13 and CeHSF18 were all up-regulated under low-temperature treatment, whereas the expression of CeHSF15 and CeHSF21 continued to decrease under low-temperature stress [42]. In Ginger, zohsf showed an expression pattern that was first up-regulated and then down-regulated under high temperature and high light stress [43]. In this study, six VbHsf genes were subjected to qRT-PCR under low-temperature conditions, and the results showed that the expression of six genes, VbHsf5, VbHsf14, VbHsf17, VbHsf18, VbHsf20, and VbHsf21, were upregulated. It was hypothesised that the up-regulated expression of these genes is important for the adaptation of V. bonariensis to low-temperature stress and the triggering of cold-response mechanism.

Conclusion

In this study, a comprehensive and systematic analysis was conducted on the Hsf gene family of V. bonariensis. A total of 25 members of the Hsf gene family were identified. The analysis encompassed gene structure, conserved motifs, conserved domains, chromosome distribution, evolutionary relationship, and distribution of cis-acting elements. The findings revealed a highly conserved structure of the Hsf gene family, which plays a crucial role in various aspects of the growth and development of V. bonariensis. The expression patterns of VbHsf genes in various tissues and organs of V. bonariensis and their responses to low-temperature stress were investigated. The results showed that the VbHsf gene family plays an important role in the development and response to low-temperature stress in V. bonariensis. We demonstrated that six genes, VbHsf5, VbHsf14, VbHsf17, VbHsf18, VbHsf20, and VbHsf21, exhibited positive regulatory roles in the response of V. bonariensis to low temperature stress. These findings provide a basis for further exploration of the functions of Hsf transcription factors in plant growth, development, and response to low temperature stress, specifically in the context of the V. bonariensis species.

Materials and methods

Plant material

V. bonariensis seeds were purchased from Benary Seed Company, Germany. Seedling conservation, seedling multiplication and hybridisation were carried out in the Seed Resource Nursery of the Key Laboratory of the Ministry of Education of Guizhou University (latitude 26°11′- 26°34′N, longitude 106°27′- 106°52′E). Two month old V. bonariensis plants in good growth condition were taken for experimental treatments.

Identification and physicochemical analysis of Hsf gene family members in V. bonariensis

First, we obtained the identified 21 A. thaliana Hsf protein sequences [44] and the existing genome data of V. bonariensis from the A. thaliana database (https://www.arabidopsis.org/). Next, we downloaded the hidden Markov model [23]of the Hsf-type DBD domain (PF00447) from the Pfam database (https://www.ebi.ac.uk/interpro/entry/pfam). We then conducted preliminary screening and identification using the HMMER software [45]. To ensure the presence of a complete Hsf domain, we compared and analyzed the obtained Hsf gene family members using tools on the NCBI CDD [46] (https://www.ncbi.nlm.nih.gov/cdd). To predict the length, molecular weight, theoretical isoelectric point, instability coefficient, fat coefficient, and hydrophilic average coefficient of the protein sequence of the Hsf gene family members in V. bonariensis, we utilized the online tool ProtParam [47] (https://web.expasy.org/protparam/). Additionally, we employed online tools, such as TMHMM2.0 (https://services.healthtech.dtu.dk/services/TMHMM-2.0/), Cell-PLoc (http://www.csbio.sjtu.edu.cn/bioinf/Cell-PLoc-2/), and SOPMA (https://npsa-prabi.ibcp.fr/cgi-bin/npsa_automat.pl?page=npsa_sopma.html), to predict the transmembrane structure characteristics, subcellular localization, and secondary structure of the Hsf gene family members in V. bonariensis.

Visualization of gene structure, domain and conserved motif of hsf gene family members in V. bonariensis

The introns and exons information of the Hsf gene family members were obtained from the genome database of V. bonariensis. Additionally, the conserved domain information of the Hsf gene family members was acquired using the CDD database (https://www.ncbi.nlm.nih.gov/Structure/cdd/wrpsb.cgi) in NCBI. To predict the conserved motif, the online tool MEME (https://meme-suite.org/meme/tools/meme) was utilized, and the visualization of the motif was done through TBtools software. For domain analysis, various software tools were employed to analyze the key conserved domains, namely DBD, OD, NLS, NES, and AHA. To understand the sequence characteristics of the DBD domain and HR-A/B region of VbHsf, the amino acid sequence was compared using MUSCLE software, and the sequence was subsequently edited using DNAMAN software [23]. The NLS domain of VbHsfs was predicted using the cNLS Mapper software [48] available at https://nls-mapper.iab.keio.ac.jp/cgi-bin/NLS_Mapper_form.cgi. For predicting the NES domain of VbHsfs, the LocNES software [49] accessible at http://prodata.swmed.edu/LocNES/LocNES.php was utilized. Lastly, the prediction of the AHA domain was based on the conserved AHA motif sequence FWxxF/L, F/I/L [50].

Phylogenetic analysis and chromosome mapping of hsf gene family members in V. bonariensis

The identified Hsf family genes of V. bonariensis were aligned with the Hsf family genes of A. thaliana using the multiple sequence alignment tool MUSCLE in MEGA software. The Max Iterations parameter was set to 100, while other parameters were left at their default values [51]. The phylogenetic tree of the Hsf family genes in V. bonariensis was constructed using the maximum likelihood method ( ML) [52]. The amino acid substitution model used was JTT + G, and the phylogenetic analysis was carried out. To obtain the support rate of each node, the bootstrap method was applied with 1000 repetitions, and the Partial deletion value was set to 80%. The resulting evolutionary tree was edited and enhanced using the online tree diagram tool iTOL [53] (https://itol.embl.de/).The location information of the Hsf gene family members was retrieved from the genome database of V. bonariensis and visualized using the TBtools software.

Analysis of cis-acting elements of hsf gene family members in V. bonariensis

The TBtools software was utilized to retrieve the promoter sequence, specifically the 2000 bp region upstream of the initiation codon, from the V. bonariensis database. The obtained promoter sequence was then subjected to analysis using the online tool PlantCARE [54] (http://bioinformatics.psb.ugent.be/webtools/plantcare/html/) to predict the cis-acting elements that can be recognized and specifically bound by transcription factors. Detailed analysis and numerical statistics were conducted, and the results were visualized using TBtools.

Colinearity analysis of hsf gene family members in V. bonariensis

The genome and annotation files of V. bonariensis were obtained from the database. The MCScanX software [55] was employed to detect gene duplication events involving the VbHsf gene. The mapping of these events was conducted using the TBtools software. To assess the selection pressure on the genes during evolution, the ratio of non-synonymous substitution rate (Ka) and synonymous substitution rate (Ks) was calculated for each non-synonymous site of the identified homologous gene pairs obtained from MCScanX.

Differences in the expression of hsf gene family members in V. bonariensis

The expression levels (FPKM) of four different parts (flowers, young stems, old stems, and leaves) and the expression levels of V. bonariensis under cold stress were retrieved from the V. bonariensis database. Subsequently, the FPKM values were visualized using TBtools.

qRT-PCR validation

Two-month-old long and healthy V. bonariensis plants were used for the cold treatment, and untreated plants with similar growth conditions were used as controls. 4 °C was used as the cold treatment condition, and after 3, 6, 9, 12, and 24 h, the V. bonariensis leaves were taken as samples. The samples were placed in sterile, enzyme-free freezing tubes and immediately frozen in liquid nitrogen, and subsequently these samples were stored at -80 °C for RNA extraction. RNA was extracted from the samples by referring to the instructions of the OMEGA Extraction Kit (OMEGA, USA), and cDNA was synthesised by reversing the sample RNAs using the StarScript III RT Kit.The cDNA was used as a template for real-time quantitative PCR assay. qRT-PCR reactions were performed using a qTOWER3G system (China) and 2×RealStar Fast SYBR qPCR Mix (GENStar, China). A total volume of 10 µL was prepared for each reaction, including 5 µL of 2×RealStar Fast SYBR qPCR Mix, 0.5 µL each of forward and reverse primers, 1 µL of diluted cDNA, and 3 µL of DEPC water. three replicates were set up for each sample to ensure the reliability of experimental data. Gene-specific primers were designed using the NCBI online website (https://www.ncbi.nlm.nih.gov/tools/primer-blast/). VbACTIN was used as an internal reference gene. Data were analysed using the 2^-ΔΔCT method [56].

Data availability

The datasets used and/or analyzed during the current study are available from the corresponding author on reasonable request.

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Acknowledgements

We express our gratitude to all the members of the laboratory at Guizhou University for their invaluable support and assistance during the entire research process.

Funding

This study is supported by the Key Project of Guizhou Basic Research Program (Guizhou Science and Science Foundation ZK [2023] Key 006), the National Natural Science Foundation of China (32160722).

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  1. Xiuliu Yang and SiSi Wang contributed equally to this work.

Authors and Affiliations

  1. Key laboratory of Plant Resource Conservation and Germplasm Innovation in Mountainous Region (Ministry of Education), College of Life Sciences, Institute of Agro-bioengineering, Guizhou University, Guiyang, 550025, Guizhou Province, China

    Xiuliu Yang, Sisi Wang, Ju Cai, Tao Zhang, Dandan Yuan & Yan Li

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  1. Xiuliu Yang
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Contributions

YL design experiment; XLY, SSW and JC conducted experimental analysis of the data; XLY and SSW edit manuscript; TZ and DDY contribution reagents/materials/analytical tools; XLY revised the manuscript. All authors read and approved the final manuscript.

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Correspondence to Yan Li.

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Yang, X., Wang, S., Cai, J. et al. Genome-wide identification, phylogeny and expression analysis of Hsf gene family in Verbena bonariensis under low-temperature stress. BMC Genomics 25, 729 (2024). https://doi.org/10.1186/s12864-024-10612-8

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