General Analysis of Heat Shock Factors in the Cymbidium ensifolium Genome Provided Insights into Their Evolution and Special Roles with Response to Temperature

Heat shock factors (HSFs) are the key regulators of heat stress responses and play pivotal roles in tissue development and the temperature-induced regulation of secondary metabolites. In order to elucidate the roles of HSFs in Cymbidium ensifolium, we conducted a genome-wide identification of CeHSF genes and predicted their functions based on their structural features and splicing patterns. Our results revealed 22 HSF family members, with each gene containing more than one intron. According to phylogenetic analysis, 59.1% of HSFs were grouped into the A subfamily, while subfamily HSFC contained only two HSFs. And the HSF gene families were differentiated evolutionarily between plant species. Two tandem repeats were found on Chr02, and two segmental duplication pairs were observed on Chr12, Chr17, and Chr19; this provided evidence for whole-genome duplication (WGD) events in C. ensifolium. The core region of the promoter in most CeHSF genes contained cis-acting elements such as AP2/ERF and bHLH, which were associated with plant growth, development, and stress responses. Except for CeHSF11, 14, and 19, each of the remaining CeHSFs contained at least one miRNA binding site. This included binding sites for miR156, miR393, and miR319, which were responsive to temperature and other stresses. The HSF gene family exhibited significant tissue specificity in both vegetative and floral organs of C. ensifolium. CeHSF13 and CeHSF15 showed relatively significant expression in flowers compared to other genes. During flower development, CeHSF15 exhibited markedly elevated expression in the early stages of flower opening, implicating critical regulatory functions in organ development and floral scent-related regulations. During the poikilothermic treatment, CeHSF14 was upregulated over 200-fold after 6 h of heat treatment. CeHSF13 and CeHSF14 showed the highest expression at 6 h of low temperature, while the expression of CeHSF15 and CeHSF21 continuously decreased at a low temperature. The expression patterns of CeHSFs further confirmed their role in responding to temperature stress. Our study may help reveal the important roles of HSFs in plant development and metabolic regulation and show insight for the further molecular design breeding of C. ensifolium.


Introduction
Cymbidium ensifolium belongs to the orchid genus Cymbidium and holds high ornamental and medicinal values [1][2][3].It serves as a potential parental line for the modern horticultural hybridization of orchids, especially in the development of heat-tolerant, earlyflowering, and fragrant hybrids [4].In recent years, abiotic stresses such as drought, high temperatures, salinity, and rising carbon dioxide have affected plant growth and agricultural development [5][6][7].Among these, high temperature is one of the biggest challenges faced worldwide [8].In the past few decades, there has been an increase in regions experiencing extremely high temperatures in China.This trend is primarily concentrated in the Yangtze River Delta region, eastern Sichuan, the Beijing-Tianjin-Hebei region, and southeastern coastal areas [9].Fujian Province, the origin of C. ensifolium, has experienced dramatic temperature changes and frequent heat events in recent decades [10].Due to various abiotic stresses, the habitats of C. ensifolium are declining, leading to the shrinking of wild populations [11,12].C. ensifolium, currently domesticated as vital ornamental orchids, has formed a global industry chain, requiring large quantities to meet market demands.Gaining a clear understanding of the heat response mechanisms of C. ensifolium is one of the key methods in modern orchid breeding.However, there has been a scarcity of relevant research.
Heat stress transcription factors (HSFs) are crucial in the plant stress response, playing pivotal roles in adapting to various abiotic stresses.During biotic or abiotic stresses, TFs bind to specific promoter regions of target genes, activating or repressing their transcription and orchestrating defensive responses [13].For instance, they regulate the expression of stress-responsive genes like heat shock proteins (HSPs) [14].HSFs possess five conserved domains: the N-terminal DNA-binding domain (DBD), oligomerization domain (OD), nuclear localization signal (NLS), nuclear export signal (NES), and C-terminal activation domain (CAD) [15,16].The CAD can modulate gene expression in response to heat shock [17].The NLS and NES regulate the nuclear import/export and distribution of HSPs.Due to the conservation of DBD and OD, they are often used to identify HSFs.They also enable binding to HSP promoters [18].Based on structural features, HSFs are classified into three subfamilies: HSFA, HSFB, and HSFC [19].These HSF subfamilies facilitate plant responses to abiotic stress by activating HSP expression [20].
Current studies have shown that HSFs respond to heat stress signals in plants and regulate growth and development [21].In Arabidopsis, salt and osmotic and cold stress can increase the expression of the stress-responsive gene HSFA6b via ABA mediation [22].HSFA was found to modulate thermotolerance and cell wall integrity in Aspergillus fumigatus [23].Overexpression of HSFA9 in Helianthus annuus promoted the accumulation of carotenoids, chlorophyll, and chloroplasts, enhancing cotyledon development [24].Under drought, heat, and salt stresses, TrHSFB2a acted as a negative regulator of stress tolerance in white clovers [25].The HSFB of Marchantia polymorpha played a pivotal role in thermal reactions, overseeing the development of meristem branching and fostering the formation of anther stems [26].AtHSFB4 overexpression caused shortened root length in Arabidopsis, impacting root development [27].Furthermore, the HSF gene exhibited a certain correlation with the formation of floral compounds in specific plants, affecting the flower development process [28][29][30].Hence, exploring the functions of HSF genes in enhancing both plant stress resistance and breeding holds paramount significance.
Since the identification of the HSF family in A. thaliana [31], increasing numbers of HSF families have been uncovered in other plants, including tomato [20], Oryza sativa [32], Secale cereale [33], Zea mays [34], Pisum sativum [35], kiwifruit [36], Salvia miltiorrhiza [37], and Glycine max [38].However, little documentation exists on this family in orchids.During the previous researches of the research group, We found that this protein has multiple functions, especially for the formation of endogenous floral substance.Here, we performed a whole identification of CeHSF genes in C. ensifolium.Through analyzing their phylogeny, gene structure, motif composition, cis elements, protein-protein interactions, miRNA targets, and expression patterns, we obtained a panoramic regulation and induction model.We also examined the expression of CeHSFs under heat and cold treatments using qRT-PCR to find out the detailed gene reponses for further usage.Our findings may establish a foundation for functional studies of HSFs in orchids.

Phylogenetic Analysis and Identification of HSFs in C. ensifolium
Based on the genome data of C. ensifolium, a total of 22 HSF genes were identified (Supplementary Table S7).A total of 21 AtHSFs, 25 OsHSFs, 18 PeHSFs, and 22 CeHSFs were used to construct a phylogenetic tree.Phylogenetic analysis showed that the 22 members were divided into three groups (Group A, Group B, and Group C) (Figure 1).This phylogenetic classification was consistent with the presence of conserved DNA-binding domains (DBDs) and the established classification of Arabidopsis HSFs.Group A had the most C. ensifolium HSF members with thirteen, while Group C had the fewest with only two.There were seven C. ensifolium HSF members in Group B. The phylogenetic tree results highlighted some differences between monocot and eudicot HSFs, such as eudicots having one clade in Group C (C1), while monocots had two (C1 and C2).
miRNA targets, and expression patterns, we obtained a panoramic regulation and induction model.We also examined the expression of CeHSFs under heat and cold treatments using qRT-PCR to find out the detailed gene reponses for further usage.Our findings may establish a foundation for functional studies of HSFs in orchids.

Phylogenetic Analysis and Identification of HSFs in C. ensifolium
Based on the genome data of C. ensifolium, a total of 22 HSF genes were identified (Supplementary Table S7).A total of 21 AtHSFs, 25 OsHSFs, 18 PeHSFs, and 22 CeHSFs were used to construct a phylogenetic tree.Phylogenetic analysis showed that the 22 members were divided into three groups (Group A, Group B, and Group C) (Figure 1).This phylogenetic classification was consistent with the presence of conserved DNAbinding domains (DBDs) and the established classification of Arabidopsis HSFs.Group A had the most C. ensifolium HSF members with thirteen, while Group C had the fewest with only two.There were seven C. ensifolium HSF members in Group B. The phylogenetic tree results highlighted some differences between monocot and eudicot HSFs, such as eudicots having one clade in Group C (C1), while monocots had two (C1 and C2).The CeHSFs were designated as CeHSF1 to CeHSF22 according to their chromosomal sequences and locations.Corresponding gene IDs, names, chromosomal loci, etc., were tabulated (Table 1).Analysis of various physicochemical parameters showed that the CeHSF proteins ranged from 190 to 514 amino acids in length, 22.4 to 51.6 kDa in molecular weight, 4.82 to 9.61 in theoretical pI, 39.1 to 72.6 in instability index, 66.7 to 83.3 in aliphatic index, and −0.899 to −0.396 in grand average hydropathicity, consistent with hydrophilic proteins (Table 1).

Gene Structures and Conserved Motifs of C. ensifolium HSFs
Based on genomic DNA sequences, the gene structures and conserved motifs of CeHSFs in C. ensifolium were analyzed.HSF proteins all contained the DBD domain (three α-helices and four β-folded layers), which was highly conserved.Sequence alignment between C. ensifolium and Arabidopsis indicated that C. ensifolium HSF proteins had the typical DBD structure (Figure 2A).In the α3-helix, all AtHSFs and CeHSFs were highly conserved, except CeHSF22, which had a 27-amino acid insertion segment in its α3-helix.Motif analysis of C. ensifolium HSFs showed that motif1, motif2, and motif4 were simultaneously present in most members, representing the major motifs constituting the HSF family (Figure 2B).Meanwhile, Groups B and C exhibited high similarity in conserved motifs, implying analogous functions for members of these two groups.However, different groups also had distinct conserved motifs, which might be related to their regulatory roles.Notably, Group C members only had 4 motifs (motifs 1, 2, 3, and 4), while Group B proteins contained motifs 1, 2, and 4, as well as motifs 5, 15, and 11.Motif 11 only was unique to Group B. Aside from the shared motifs, Group A also possessed motifs 6, 7, 8, 9, 10, 12, 13, and 14.
Exon-intron analysis uncovered exon numbers ranging from two to five, among the 22 CeHSF genes (Figure 2C).CeHSF6 had the maximal five exons, while over half of the CeHSF genes (13, 59.1%) contained two exons.All CeHSFs exhibited one to four introns, with 13 genes harboring just one intron similar to the exon pattern.CeHSF6 contained the most introns at four, implying greater functional diversity.Approximately 72.7% of the 22 CeHSF genes lacked UTRs, while CeHSF18 had the most UTRs at three.

Secondary and Tertiary Structures of the CeHSF Proteins
The HSF protein's secondary structure included an alpha helix, an extended strand, a beta turn, and a random coil.The secondary structure of the 22 CeHSF proteins was analyzed using SOPMA (Supplementary Table S1; Figure 3B).The results showed that alpha helices and random coils were the main secondary structural elements (alpha helix 34.27-56.63%,random coil 31.49-51.05%),followed by extended chains and folding (extended strand 7.18-10.14%,beta turn 4.55-6.85%).Similar results were found for the rest of the proteins (Supplementary Table S1).
The protein sequences were submitted to Alphafold2 online tool for tertiary structure prediction analysis (Supplementary Table S2).We selected three proteins with a certain degree of conservation to display the tertiary structures (Figure 3A).The secondary and tertiary structure results showed that CeHSF2, CeHSF3, and CeHSF22 had similar ratios of alpha helices, extended strands, beta turns, and random coils.The random coil ratio of CeHSF22 was markedly higher than that of other HSF proteins.The tertiary structure of the HSF protein was mainly composed of alpha helices, and the tertiary structures of CeHSF2, CeHSF3, and CeHSF22 were similar.
Gene duplication events, including tandem and segmental duplications, play vital roles in gene duplication and neofunctionalization. Analysis of duplication events in CeHSFs identified two tandem repeats on Chr02 and two segmental duplication pairs between Chr12 and Chr17 and between Chr12 and Chr19 (Figure 4).Integrated with phylogenetic analysis, genes involved in tandem and segmental duplications clustered together on the phylogenetic tree, implying expansion of these CeHSFs during evolution.Overall, these results indicated that most CeHSFs were relatively conserved, except for the duplication events on Chr02, Chr12, Chr17, and Chr19.S1 and S2.
Gene duplication events, including tandem and segmental duplications, play vital roles in gene duplication and neofunctionalization. Analysis of duplication events in CeHSFs identified two tandem repeats on Chr02 and two segmental duplication pairs between Chr12 and Chr17 and between Chr12 and Chr19 (Figure 4).Integrated with phylogenetic analysis, genes involved in tandem and segmental duplications clustered together on the phylogenetic tree, implying expansion of these CeHSFs during evolution.Overall, these results indicated that most CeHSFs were relatively conserved, except for the duplication events on Chr02, Chr12, Chr17, and Chr19.

Protein-Protein Interaction Network Analysis of CeHSF Family Members
Protein-protein interaction (PPI) prediction was performed to gain further insight into the biological roles and regulatory networks of CeHSFs.A total of 51 proteins were detected, including 11 CeHSFs, that exhibited interactions, with 22 proteins interacting with CeHSFs (Figure 5A, Supplementary Table S3).Most proteins interacting with CeHSFs were functionally validated as related to heat stress, such as HSP90-1, HSP70-4, HSBP, and HSFA1A.Additionally, proteins involved in plant development or non-biotic stress responses, like MPK3, MPK6, ZAT6, FKBP62, and FKBP65, also interacted with CeHSFs.PPI network prediction further revealed potential interactions between different CeHSFs.Among the 11 CeHSFs, interactions were found among CeHSF2, CeHSF7, CeHSF10, CeHSF12, and CeHSF14 (Figure 5B), implying that CeHSFs may form complexes in response to stress.

Protein-Protein Interaction Network Analysis of CeHSF Family Members
Protein-protein interaction (PPI) prediction was performed to gain further insig into the biological roles and regulatory networks of CeHSFs.A total of 51 proteins we detected, including 11 CeHSFs, that exhibited interactions, with 22 proteins interacti with CeHSFs (Figure 5A, Supplementary Table S3).Most proteins interacting w CeHSFs were functionally validated as related to heat stress, such as HSP90-1, HSP70 HSBP, and HSFA1A.Additionally, proteins involved in plant development or non-bio stress responses, like MPK3, MPK6, ZAT6, FKBP62, and FKBP65, also interacted w

Cis-Acting Elements in the Promoter Regions of CeHSF Genes
To understand the genetic functions and regulatory mechanisms of CeHSF genes, the cis-regulatory elements from CeHSF genes' upstream promoter regions were predicted through the PlantPAN website.Thirty-six types of binding sites of transcription factor families were identified within 2000 bp upstream of the promoter in 22 CeHSF genes.These 36 categories appeared 12,297 times in the promoter regions of 22 CeHSF genes, among which AP2/ERF, AT-Hook, and GATA elements were the most prevalent (Figure 6A).The core promoter region of most CeHSF genes contained elements such as AP2/ERF, bHLH, bZIP, Dof, GATA, MYB, NF-YB/NF-YA/NF-YC, and ZF-HD.In addition, we also divided the promoter region into three sub-regions (1-500 kb, 501-100 kb, and 1001-2000 kb) (Figure 6B-D).We observed that AP2/ERF appeared 266 times in CeHSF18, accounting for 45.24% of the total number of loci in the gene.It was also the most abundant region among the 22 CeHSF genes, indicating that this gene might have been involved in plant growth, development, and stress responses.In addition, it is noteworthy that the LFY was only found in CeHSF12 and the LOB/LBD was only found in CeHSF11, which means they might have been involved in cell proliferation, floral organ development, and nitrogen metabolism regulation.Overall, most genes had at least 20 different binding sites in the core region of the promoter, which were implicated in plant growth metabolism (AT Hook, AP2/ERF) and abiotic stress (bZIP, Dehydrin).

Cis-Acting Elements in the Promoter Regions of CeHSF Genes
To understand the genetic functions and regulatory mechanisms of CeHSF genes, the cis-regulatory elements from CeHSF genes' upstream promoter regions were predicted through the PlantPAN website.Thirty-six types of binding sites of transcription factor families were identified within 2000 bp upstream of the promoter in 22 CeHSF genes.These 36 categories appeared 12,297 times in the promoter regions of 22 CeHSF genes, among which AP2/ERF, AT-Hook, and GATA elements were the most prevalent (Figure 6A).The core promoter region of most CeHSF genes contained elements such as AP2/ERF, bHLH, bZIP, Dof, GATA, MYB, NF-YB/NF-YA/NF-YC, and ZF-HD.In addition, we also divided the promoter region into three sub-regions (1-500 kb, 501-100 kb, and 1001-2000 kb) (Figure 6B-D).We observed that AP2/ERF appeared 266 times in CeHSF18, accounting for 45.24% of the total number of loci in the gene.It was also the most abundant region among the 22 CeHSF genes, indicating that this gene might have been involved in plant growth, development, and stress responses.In addition, it is noteworthy that the LFY was only found in CeHSF12 and the LOB/LBD was only found in CeHSF11, which means they might have been involved in cell proliferation, floral organ development, and nitrogen metabolism regulation.Overall, most genes had at least 20 different binding sites in the core region of the promoter, which were implicated in plant growth metabolism (AT Hook, AP2/ERF) and abiotic stress (bZIP, Dehydrin).

Excavating miRNA Targets for CeHSF Genes
We selected 22 members of the CeHSF gene family as candidate target gene sequences to predict their miRNAs.The results (Figure 7; Supplementary Table S4) showed that except for the CeHSF11, CeHSF14, and CeHSF19 members CeHSFs, other CeHSF family genes were predicted targets of at least one miRNA.CeHSF18 was one of the most targeted HSF genes, predicted to be targeted by seven miRNAs.Most genes had at least one or two targeting binding sites of miRNAs (Figure 7).CeHSF2, 3, and 6 were predicted to contain miR156 binding sites, a temperature-sensitive miRNA that coordinates plant flowering in response to temperature.Additionally, several miRNA binding sites related to plant defense mechanisms were identified, including sites for miR393 and miR319.These findings implicated the existence of a complex network regulation system between miRNA and CeHSFs.

Excavating miRNA Targets for CeHSF Genes
We selected 22 members of the CeHSF gene family as candidate target gene sequences to predict their miRNAs.The results (Figure 7; Supplementary Table S4) showed that except for the CeHSF11, CeHSF14, and CeHSF19 members of CeHSFs, other CeHSF family genes were predicted targets of at least one miRNA.CeHSF18 was one of the most targeted HSF genes, predicted to be targeted by seven miRNAs.Most genes had at least one or two targeting binding sites of miRNAs (Figure 7).CeHSF2, 3, and 6 were predicted to contain miR156 binding sites, a temperature-sensitive miRNA that coordinates plant flowering in response to temperature.Additionally, several miRNA binding sites related to plant defense mechanisms were identified, including sites for miR393 and miR319.These findings implicated the existence of a complex network regulation system between miRNA and CeHSFs.

Expression Profiles of HSF Genes in C. ensifolium Tissues and Development Stages
Based on transcriptomic data, the expression patterns of 22 HSF genes in C. ensifolium are shown in Figure 8.Among all 22 CeHSF genes, the expression levels of CeHSF1, CeHSF2, CeHSF3, CeHSF4, CeHSF5, CeHSF6, CeHSF10, and CeHSF22 genes were very low in all samples, below detection limits.However, no gene showed significantly high expression in all samples.The HSF gene family exhibited certain tissue specificity in both vegetative and floral organs of C. ensifolium (Figure 8A,B).Among them, CeHSF13 and CeHSF15 showed relatively significant expression in flowers compared to other genes.Additionally, these two genes exhibited the highest expression levels in the gynandrium.The expression of CeHSF11, CeHSF13, CeHSF15, CeHSF18, and CeHSF21 in vegetative organs was relatively higher.Specifically, CeHSF13 displayed remarkably significant expression in roots, CeHSF15 exhibited the most prominent expression in leaves, and CeHSF21 showed the most significant expression in pseudobulbs.It is noteworthy that HSF genes exhibited certain expression patterns during the growth and development of flowers.For instance, in the process of floral bud development, the expression levels of CeHSF7, CeHSF14, CeHSF18, and CeHSF21 showed an increasing trend (Figure 8C).During different developmental stages of flowers, the expression level of CeHSF12 gradually increased, reaching its highest level during the peak flowering stage, and then gradually decreased as the flower deteriorated.CeHSF15 also showed a similar trend of initially increasing and then decreasing expression, with the most significant expression occurring during the early opening stage (Figure 8D).Meanwhile, CeHSF13 exhibited a trend of initially decreasing and then increasing expression.Its expression level decreased as the flower bud matured, and then gradually increased as the flower opened (Figure 8D).It is worth noting that HSF genes presented certain expression patterns during the growth and development of flowers.For instance, during the process of floral bud development, the expression levels of CeHSF7, CeHSF14, CeHSF18, and CeHSF21 showed an increasing trend (Figure 8C).Throughout different developmental stages of flowers, the expression level of CeHSF12 gradually increased, reaching its highest level during the peak flowering stage, and then gradually decreased as the flower deteriorated.CeHSF15 also exhibited a similar trend of initially increasing and then decreasing expression, with the most significant expression occurring during the initial opening stage (Figure 8D).Meanwhile, CeHSF13 displayed a trend of initially decreasing and then increasing expression.Its expression level decreased as the flower bud matured, and then gradually increased as the flower opened (Figure 8D).S4.

Expression Profiles of HSF Genes in C. ensifolium Tissues and Development Stages
Based on transcriptomic data, the expression patterns of 22 HSF genes in C. ensifolium are shown in Figure 8.Among all 22 CeHSF genes, the expression levels of CeHSF1, CeHSF2, CeHSF3, CeHSF4, CeHSF5, CeHSF6, CeHSF10, and CeHSF22 genes were very low in all samples, below detection limits.However, no gene showed significantly high expression in all samples.The HSF gene family exhibited certain tissue specificity in both  S4.
peak flowering stage, and then gradually decreased as the flower deteriorated.CeHSF15 also exhibited a similar trend of initially increasing and then decreasing expression, with the most significant expression occurring during the initial opening stage (Figure 8D).Meanwhile, CeHSF13 displayed a trend of initially decreasing and then increasing expression.Its expression level decreased as the flower bud matured, and then gradually increased as the flower opened (Figure 8D).

Expression of CeHSFs in Response to Different Temperature Treatments
Seven genes with relatively higher expression in roots, pseudobulbs, leaves, bracts, and different flower development stages and other parts were selected as target genes.These genes were most likely to respond to the regulation of expression under temperature stress (Figure 9).The results of high-temperature treatment showed that the expression of CeHSF18 was sharply induced and remained highly expressed.CeHSF7, CeHSF14, CeHSF15, and CeHSF21 exhibited a consistent pattern, with significant up-regulation after 6 h of high-temperature treatment, rapid down-regulation after 12 h, and elevated expression again after 24 h.Interestingly, CeHSF14 showed a nearly 200-fold up-regulation after 6 h of high-temperature treatment (Figure 9).At 12 h of high-temperature treatment, the expression of CeHSF11 and CeHSF13 was continuously inhibited.But after 24 h, their expression was higher than that before high-temperature treatment.The results of lowtemperature treatment showed a similar pattern for CeHSF7, CeHSF11, CeHSF13, CeHSF14, and CeHSF18.CeHSF7, CeHSF11, and CeHSF18 were highly expressed at 12 h of low temperature, with a decrease in expression after 24 h.CeHSF13 and CeHSF14 showed the highest expression at 6 h of low temperature, followed by down-regulation.In the low-temperature environment, the expression of CeHSF15 and CeHSF21 continuously decreased.Gene expression was significantly inhibited.Interestingly, CeHSF15 exhibited repaid induction and stepwise suppression during cold temprature, this inferred opposite models of regulation.Combining the transcriptome data, CeHSF15 expressed earlier than the presence of floral scents.In the process of flowering, CeHSF15 may act as a trigger in the lanch of flower scents.
regulation after 6 h of high-temperature treatment (Figure 9).At 12 h of high-temperature treatment, the expression of CeHSF11 and CeHSF13 was continuously inhibited.But after 24 h, their expression was higher than that before high-temperature treatment.The results of low-temperature treatment showed a similar pattern for CeHSF7, CeHSF11, CeHSF13, CeHSF14, and CeHSF18.CeHSF7, CeHSF11, and CeHSF18 were highly expressed at 12 h of low temperature, with a decrease in expression after 24 h.CeHSF13 and CeHSF14 showed the highest expression at 6 h of low temperature, followed by down-regulation.In the low-temperature environment, the expression of CeHSF15 and CeHSF21 continuously decreased.Gene expression was significantly inhibited.Interestingly, CeHSF15 exhibited repaid induction and stepwise suppression during cold temprature, this inferred opposite models of regulation.Combining the transcriptome data, CeHSF15 expressed earlier than the presence of floral scents.In the process of flowering, CeHSF15 may act as a trigger in the lanch of flower scents.
Further correlation analysis indicates a highly significant correlation between CeHSF14, CeHSF7, CeHSF15, and CeHSF21.The expression of these four genes may have a certain relationship.CeHSF11 was significantly correlated with CeHSF13, suggesting a potential relationship in the expression of these two genes (Figure 9).Further correlation analysis indicates a highly significant correlation between CeHSF14, CeHSF7, CeHSF15, and CeHSF21.The expression of these four genes may have a certain relationship.CeHSF11 was significantly correlated with CeHSF13, suggesting a potential relationship in the expression of these two genes (Figure 9).

Conservation and Expansion of CeHSF Family Members
HSF genes play a crucial role in various aspects of plant growth, development, and response to different stresses, including salt, heat, and cold stress [39,40].Therefore, diversity in HSF family members has long been a subject of considerable attention.Currently, HSF genes have been identified in numerous plant species, including Brassica napus (64 members) [41], Populus trichocarpa (28 members), and Medicago truncatula (16 members) [42].The number of members in the HSF family varies in different Gossypium species, ranging from 31 to 78 [43].Similarly, monocotyledonous plants such as Triticum aestivum, rye, rice, and maize have 82, 31, 22, and 25 identified members [32][33][34]44], respectively.Discrepancies in the number of HSF family members among different plants may be attributed to the differential retention of HSF genes during the evolutionary process to adapt to the environment.In this study, 22 HSF gene family members were identified in C. ensifolium, a number similar to some monocot plants such as rice and maize [32,34] (Figure 1).CeHSFs displayed a high degree of conservation in their sequences (Figure 2A).However, some duplication was observed in the A2 and B4 subfamilies (Figure 1, Figure 4).The diversity of HSF family members among different plants might be linked to whole-genome duplication (WGD) events in plants, as seen in the differences between members in A. thaliana and soybean [45,46].C. ensifolium has undergone WGD events in its evolutionary history [47], which might explain the expansion of CeHSF family members.

Functional Prediction and Abiotic Stress Response of CeHSFs
Extreme environments currently threaten plant survival and development worldwide.MicroRNAs (miRNAs) are short non-coding RNAs that have emerged as key post-transcriptional regulators in many species.Under extreme conditions, miRNAs are one of the mechanisms for plant stress responses [48,49].Previous studies have shown miRNA involvement in abiotic stresses including drought, salinity, and temperature [48,50,51].Our investigation revealed that, with the exception of CeHSF11, 14, and 19, every other member within the CeHSF family harbored a minimum of one miRNA binding site (Supplementary Table S4).CeHSF2, 3, and 6 were predicted to contain miR156, a temperature-sensitive miRNA that coordinates plant flowering in response to temperature [51].Additionally, many miRNA binding sites associated with plant defense mechanisms were present, such as miR393 and miR319 [52].These results indicate the important roles of CeHSFs in responding to stresses, especially temperature stresses.Similar to previous studies [33,53], protein-protein interaction network analysis further revealed interactions of CeHSFs with heat stress-related proteins such as HSP70-4.
Plants can respond to environmental stresses and regulate growth via tissue-specific gene expression [54,55].Previous reports indicated differential expression of HSF genes in some species [56,57].In C. ensifolium, HSF genes showed specific expression patterns in different tissues (Figure 8).For instance, CeHSF13 was significantly expressed in roots and bracts, while CeHSF15 exhibited higher expression in leaves.Both were specifically expressed in flowers.Notably, during the growth of floral buds, the expression of CeHSF13 gradually decreased, whereas CeHSF15 showed an initial increase followed by a decrease during the transition from floral bud to open flower.Moreover, several other CeHSF genes showed significant expression, indicating that CeHSFs could have important roles in the growth and development of multiple organs and tissues in C. ensifolium.
In previous studies, HSFs have been shown to play major roles in plants' responses to abiotic stress, enhancing their thermotolerance and salt stress tolerance [16,58,59].In Dianthus caryophyllus, the majority of DcaHSFs were responsive to heat stress, while some genes were downregulated by cold stress [60].HSF genes in Hypericum perforatum showed pronounced up-regulation under heat stress [61].In Zingiber officinale, ZoHSFs exhibited an expression pattern of initial upregulation followed by down-regulation under high temperature and strong light stress [62].In this study, seven CeHSF genes were treated with high-and low-temperature treatments.The qRT-PCR results under high-temperature conditions showed the rapid up-regulation of CeHSF18 expression, while CeHSF14 exhibited significant up-regulation after 6 h of high-temperature treatment (Figure 9).Additionally, many genes showed varying degrees of expression, consistent with previous research [63], suggesting the involvement of CeHSF genes in the heat stress tolerance of C. ensifolium.Under low-temperature treatment, CeHSF7, CeHSF11, CeHSF14, CeHSF13, and CeHSF18 all exhibited up-regulation, indicating their potential role in regulating the plant response to low-temperature stress (Figure 9).However, the expression of CeHSF15 and CeHSF21 continuously decreased during low-temperature stress, suggesting that C. ensifolium may mitigate cold damage by reducing the expression of heat stress transcription factors.

Identification and Physicochemical Properties of HSF Genes in C. ensifolium Genome
The genomic sequence and annotation data for C. ensifolium [47] were downloaded from the National Genome Data Center (NGDC) (https://ngdc.cncb.ac.cn/, accessed on 22 October 2023).The HSF protein sequences of Arabidopsis were downloaded from TAIR (http://www.arabidopsis.org/,accessed on 22 October 2023).The HSF protein sequences of Arabidopsis were used as query sequences to execute a BLASTP search against the C. ensifolium genome (E-value < 1 × 10 −5 , Num of Hits: 500, Num of Aligns: 250).The BLASTP results are shown in Supplementary Table S7.The Hidden Markov model (HMM) with PF00447 (HSF-type DBD domain) was used to match the HSF gene sequences in C. ensifolium through the TBtools (version 2.019) [64] software and domain checking was performed to remove sequences without DBD domains.ProtParam online analytical tools (https://web.expasy.org/protparam/,accessed on 23 October 2023) were used to predict the number of amino acids, molecular weight, theoretical pI, instability index, aliphatic index, and grand average of hydropathicity.

Protein Interactions and Chromosome Distribution Analysis of the CeHSF Genes
Protein-protein interaction network prediction analysis was conducted using the STRING database and Cytoscape (version 3.10) [67].The 22 CeHSF protein sequences were submitted to the STRING database (http://string-db.org/cgi,accessed on 28 October 2023) using Arabidopsis orthologs as references, with no more than 20 interactors (1st and 2nd shell).All CeHSF genes were mapped to locations on different C. ensifolium chromosomes using the TBtools software (version 2.019).The syntenic relationship between the genes and replication events was analyzed by the Run MCScanx Wrapper function in TBtools (version 2.019) and visualized by Excel2021 and TBtools software (version 2.019).

Promoter Capture and Prediction of Specific miRNA targets in the CeHSF Family Members
TBtools (version 2.019) was used to obtain a 2000 bp sequence upstream of the HSF genes in C. ensifolium from the start codon.Cis-acting elements in the promoter region of the CeHSF were analyzed using PlantPAN4.0(http://plantpan.itps.ncku.edu.tw/plantpan4,accessed on 29 October 2023) [68].The data were analyzed and visualized by Excel2021.Bioinformatics and prediction analyses of miRNAs and their target CeHSF genes were performed in the web-based psRNA Target Server (https://www.zhaolab.org/psRNATarget/analysis, accessed on 29 October 2023).The expected value was set to 4.5 and the remaining parameters were set to default.Finally, alignment of identified genes with the miRNAs of A. thaliana was conducted.

Analysis of Gene Expression Patterns
In order to investigate the potential involvement of HSF genes in various organs of C. ensifolium, we downloaded the RNA-seq data of C. ensifolium from the National Genomics Data Center.The FPKM values of different CeHSF genes and the Genome Sequence Archive (GSA) RUN accession numbers were presented in Supplementary Table S5.Subsequently, RNA-Seq reads were mapped to the C. ensifolium CDS files, and gene expression was calculated using kallisto (version 0.48.0) with default parameters [70,71].The gene expression patterns were visualized using TBtools (version 2.019).

The Assay of qRT-PCR during the High-and Low-Temperature Induction
The materials for this experiment were obtained from the cultivation of the C. ensifolium variety 'Xiao Tao Hong' at the germplasm resource nursery of Fujian Agriculture and Forestry University (26 • 04 ′ 51.3 ′′ N, 119 • 14 ′ 19.9 ′′ E).To ensure the consistency of the experiment and the stability and repeatability of the results, independent plants were selected from the nursery.Four individuals were prepared as biological replicates for each treatment.Untreated plants with similar growth status were used as controls.Plants were subjected to stress by high and low temperatures, and tender leaves from the plant tops were collected at corresponding time points.The treatment involved high-temperature stress at 42 • C and low-temperature stress at 4 • C in incubators.Tender leaves of the orchid 'Xiao Tao Hong' were sampled after 6, 12, and 24 h of high-and low-temperature treatments.The samples were placed in 1.5 mL sterile non-enzymatic cryopreservation tubes and rapidly frozen in liquid nitrogen.Finally, total RNA was extracted by using the R6827 Plant RNA Kit (Omega Bio-Tek, Guangzhou, China).DNA digestion was performed to remove DNA from the total RNA extracts.The Hieff UNICON Universal Blue qPCR SYBR Green Master Mix kit (Yeasen Biotechnology, Shanghai, China) was used for reverse transcription to synthesize quantitative cDNA single strands from 2 mg RNA.This cDNA served as the template for real-time quantitative PCR detection.The CeTUB gene was used as the internal reference using fluorescence quantification.The gene sequences and internal reference primers used in the reaction are shown in Supplementary Table S6.The reaction was designed with three technical replicates.The reaction system totaled 20 µL, comprising 10 µL of Hieff UNICON Universal Blue qPCR SYBR Green Master Mix, 0.4 µL of forward primer (10 µM), 0.4 µL of reverse primer (10 µM), 4 µL of template DNA, and 5.2 µL of sterile ultrapure water.The amplification program consisted of a pre-denaturation at 95 • C for 2 min, followed by 40 cycles of denaturation at 95 • C for 10 s and annealing/extension at 60 • C for 30 s. Data were calculated using the 2 −∆∆Ct method to determine the relative gene expression levels.A one-way analysis of variance (ANOVA) was applied for data processing.Statistical differences were compared using t-tests based on IBM SPSS Statistics 24, with p < 0.05 as * and p < 0.01 as **.Finally, visual analysis was performed using Origin and Chiplot (https://www.chiplot.online/,accessed on 15 November 2023).

Conclusions
In this study, 22 CeHSFs were classified into three groups.Tandem and segmental duplications among CeHSFs represented the major expansion of this gene family.Examination of protein interaction networks, promoter cis-acting elements, and miRNA-splicing sites provided insights into the intricate stress response regulation mediated by CeHSFs.
Expression profiling across tissues suggested potential regulatory roles of CeHSFs in orchid development.High and low temperature stress experiments further demonstrated the significance of CeHSFs in modulating C. ensifolium's responses to various abiotic stimuli.CeHSF14 was shown to be the candidate TF responding to high-and low-temperature stress through possible miRNA control.
These results further underscore the significant role of HSF genes in the plant response to abiotic stress.Given the vast consumer market for C. ensifolium, breeding new varieties with strong stress resistance is of paramount importance.Therefore, studying CeHSFs will contribute to advancing breeding efforts in this regard.

Figure 1 .
Figure 1.Phylogenetic analysis of HSF genes in Cymbidium ensifolium.The neighbor-joining (NJ) phylogenetic tree was constructed using PhyloSuite with 1000 bootstraps.Group A, Group B, and

Figure 1 .
Figure 1.Phylogenetic analysis of HSF genes in Cymbidium ensifolium.The neighbor-joining (NJ) phylogenetic tree was constructed using PhyloSuite with 1000 bootstraps.Group A, Group B, and Group C were grouped together as indicated in green, red, and blue, respectively.Different colored circles represent different species; red: C. ensifolium, green: A. thaliana, blue: O. sativa, and dark blue: Passiflora edulis.

Figure 2 .
Figure 2. Multiple sequence alignment, gene structures, and conserved motif analysis in CeHSFs.(A) Multiple sequence alignment of the DBD domains among AtHSFs and CeHSFs.Red box and white character represent high homology.Red character or black bold character represent similarity within a group (column).Blue frame represents similarity across groups (columns).(B) Phylogenetic relationships and motif compositions of Cymbidium ensifolium HSF proteins.(C) Statistical analysis of introns, exons, CDSs, and UTRs in CeHSFs.

Figure 2 .
Figure 2. Multiple sequence alignment, gene structures, and conserved motif analysis in CeHSFs.(A) Multiple sequence alignment of the DBD domains among AtHSFs and CeHSFs.Red box and white character represent high homology.Red character or black bold character represent similarity within a group (column).Blue frame represents similarity across groups (columns).(B) Phylogenetic relationships and motif compositions of Cymbidium ensifolium HSF proteins.(C) Statistical analysis of introns, exons, CDSs, and UTRs in CeHSFs.

Figure 3 .
Figure 3.The prediction protein structures of CeHSF proteins.(A) Tertiary structure of 3 CeHSF proteins.(B) Secondary structure ratio of 22 CeHSF proteins.The complete secondary and tertiary structures of 22 CeHSF proteins are displayed in Supplementary TablesS1 and S2.

Figure 3 .
Figure 3.The prediction protein structures of CeHSF proteins.(A) Tertiary structure of 3 CeHSF proteins.(B) Secondary structure ratio of 22 CeHSF proteins.The complete secondary and tertiary structures of 22 CeHSF proteins are displayed in Supplementary TablesS1 and S2.

Figure 4 .
Figure 4. Chromosome localization and synteny analysis of CeHSF genes.Genes with tand repeats were linked externally with red lines, and genes with segment repeats were linked interna with black lines.a: The blue line represents gene density.b: Line colors indicate gene density Chr01-Chr20 represents the twenty chromosomes of Cymbidium ensifolium.

Figure 4 .
Figure 4. Chromosome localization and synteny analysis of CeHSF genes.Genes with tandem repeats were linked externally with red lines, and genes with segment repeats were linked internally with black lines.a: The blue line represents gene density.b: Line colors indicate gene density.c: Chr01-Chr20 represents the twenty chromosomes of Cymbidium ensifolium.

Figure 5 .
Figure 5. Predicted protein-protein interaction networks of CeHSF proteins.(A) Protein interaction networks of CeHSF proteins with other proteins.The outer circle represents proteins that interact with CeHSFs.The inner circle represents CeHSF proteins.The interaction between these proteins was represented by the gray lines.Darker colors corresponded to stronger interactions between proteins.(B) Protein interaction networks of only CeHSF proteins.

Figure 5 .
Figure 5. Predicted protein-protein interaction networks of CeHSF proteins.(A) Protein interaction networks of CeHSF proteins with other proteins.The outer circle represents proteins that interact with CeHSFs.The inner circle represents CeHSF proteins.The interaction between these proteins was represented by the gray lines.Darker colors corresponded to stronger interactions between proteins.(B) Protein interaction networks of only CeHSF proteins.

Figure 6 .
Figure 6.Analysis of cis-acting elements in CeHSF promoters.(A) Heatmap analysis of cis-acting elements of 2 kb promoter regions of CeHsf genes.(B) Cis-acting elements with starting positions from 1 to 500 kb.(C) Cis-acting elements with starting positions from 501 to 1000 kb.(D) Cis-acting elements with starting positions from 1001 to 2000 kb.

Figure 6 .
Figure 6.Analysis of cis-acting elements in CeHSF promoters.(A) Heatmap analysis of cis-acting elements of 2 kb promoter regions of CeHsf genes.(B) Cis-acting elements with starting positions from 1 to 500 kb.(C) Cis-acting elements with starting positions from 501 to 1000 kb.(D) Cis-acting elements with starting positions from 1001 to 2000 kb.

Figure 7 .
Figure 7. Prediction of targets for some microRNAs.This view displays partial regulatory roles of miRNA and CeHSF transcription factors.Blue parts show the coding region of CeHSFs.Red lines mark the splicing sites.The complete results are displayed in Supplementary TableS4.

Figure 7 .
Figure 7. Prediction of targets for some microRNAs.This view displays partial regulatory roles of miRNA and CeHSF transcription factors.Blue parts show the coding region of CeHSFs.Red lines mark the splicing sites.The complete results are displayed in Supplementary TableS4.

Figure 9 .
Figure 9. Expression analysis of the 7 CeHSF genes in leaves under different abiotic stresses (heat and cold treatments).Heatmap in the middle shows the correlation of gene expressions between the

Figure 9 .
Figure 9. Expression analysis of the 7 CeHSF genes in leaves under different abiotic stresses (heat and cold treatments).Heatmap in the middle shows the correlation of gene expressions between the two treatments.Data are means ± SE of three separate measurements based on t-test, taking p < 0.05 as * and p < 0.01 as **.

Table 1 .
Protein information of HSF gene family in Cymbidium ensifolium, including gene ID, gene name, genomics position, and protein aphysicochemical properties.