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Article

Genome-Wide Identification of Heat Shock Transcription Factor Family and Key Members Response Analysis to Heat Stress in Loquat

by
Chaojun Deng
1,2,†,
Yongping Chen
1,2,†,
Weilin Wei
1,2,
Xiuping Chen
1,2 and
Jimou Jiang
1,2,*
1
Fruit Research Institute, Fujian Academy of Agricultural Science, Fuzhou 350013, China
2
Fujian Breeding Engineering Technology Center for Longan and Loquat, Fuzhou 350013, China
*
Author to whom correspondence should be addressed.
†
These authors contributed equally to this work.
Horticulturae 2024, 10(11), 1195; https://doi.org/10.3390/horticulturae10111195
Submission received: 21 October 2024 / Revised: 6 November 2024 / Accepted: 11 November 2024 / Published: 13 November 2024
(This article belongs to the Special Issue Molecular Regulation and Maintaining of Fruit Quality)
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Abstract

:
Eriobotrya japonica (loquat) is an evergreen fruit tree of the apple tribe in Rosaceae with high edible and medicinal value. The yield and quality of loquat fruits are significantly influenced by environmental stress, particularly heat stress during fruit ripening. In this study, thirty EjHSFs were identified in the loquat genome. Twenty-nine EjHSFs were unevenly distributed across sixteen chromosomes, except Chr-6. A synteny analysis revealed that twenty-six EjHSF genes had undergone duplication events. Twenty-nine EjHSF genes were found to be in sync with HSF in apples while also diverging with other Rosaceae species. A phylogenetic analysis revealed that EjHSFs could be divided into three categories, including eighteen EjHSF-A, ten EjHSF-B, and two EjHSF-C. Twenty-nine members of the EjHSF family showed high homology to those of Malus domestica and Gillenia trifoliate. A promoter analysis retrieved thirty-three CAREs that were functionally relevant and connected to the expression of HSFs. Among these, the promoters of twenty-three EjHSF genes possessed at least one STRE element that could be activated by heat shock. Eleven of these EjHSFs were highly expressed in loquat tissues, namely EjHSF-B4a, EjHSF-A4a, EjHSF-A4d, and EjHSF-C1a in roots; EjHSF-B4b in roots and inflorescence; EjHSF-C1b in stems and roots; EjHSF-A2a in three tissues; EjHSF-A8b in four tissues; and EjHSF-A4c, EjHSF-B1a, and EjHSF-B2b in six tissues. Moreover, fifteen EjHSFs were differentially expressed (eleven upregulated and four downregulated) in fruits under heat stress treatment in the color-changing period. Among them, EjHSF-A2a and EjHSF-A2b upregulated transcriptional abundance over 300 times after heat treatment; EjHSF-B2b consistently displayed an increasing trend over time; and EjHSF-B1a was significantly downregulated. Hence, these results suggest that EjHSFs take part in loquat tissue development and may be involved in the fruit’s heat stress response. These findings enhance the understanding of EjHSFs’ role within loquats and the heat stress response of its fruit and provide target genes for heat stress improvement.

1. Introduction

Loquat (Eriobotrya japonica), belonging to the Rosaceae family, is widely cultivated in Southern China for its delicious fruit and medicinally valuable leaves [1]. The growth of loquat fruit is sensitive to fluctuations in various abiotic and biological stresses [2]. Previous studies have demonstrated that heat stress during the color-changing period significantly impacts both the yield and quality of loquat fruit. Metabolomic, transcriptomic, and small RNA analyses investigated loquat fruit in response to heat stress revealing the involvement of 36 hormone types that are crucial for fruit heat tolerance [3]. Furthermore, a differential protein analysis revealed 26 proteins associated with the defense response, carbohydrate and energy metabolism, photosynthesis, and other functional categories. Additionally, five miRNAs exhibited responsiveness to high temperatures in loquat fruits [4].
Heat shock transcription factors (HSFs) are known regulators of HSP expression and confer thermotolerance [5]. These HSFs possess several conserved functional domains, including an N-terminal deoxyribonucleic acid-binding domain (DBD), an oligomerization structural domain (OD), a nuclear localization signaling domain (NLS), a nuclear export signaling domain (NES), and a C-terminal transcriptional activation domain (CAD) [6,7]. The DBD specifically binds to heat stress elements located within the target promoter, and the OD region connects with the DBD through a hydrophobic amino acid sequence known as the HR-A/B region [8]. Furthermore, the NLS and NES play assembling roles in the nuclear import complex involving the target protein and receptor-mediated export complex with NES receptor exportin-α [9]. AHA motifs, located in the CAD of HSF plants, play a crucial role in their activator function and exhibit specificity toward HSFA-specific motifs. HSFs are classified into three families based on the length of the flexible linker region between the DBD and HR-A/B region and the number of amino acid residues: subfamilies A (A1–9), B (B1–4), and C (C1–2) [10].
The comprehensive identification of HSF has been accomplished in various plant species by developing and utilizing genomic data. For instance, fifty-six HSFs have been identified to respond to heat stress and other biotic stresses in wheat (Triticum aestivum) [11]. Several HSFs have been identified in various plants since the first HSF was identified in tomatoes and functions associated with heat stress. TrHSFB2a of white clover (Trifolium repens) negatively regulates drought, heat, and salt stress tolerance in transgenic Arabidopsis [12]. Overexpression of PsnHSF21 confers salt tolerance in Populus simonii × P. nigra trees [13]. Moreover, AtHOPs participate in folding and stabilizing HSFA1a alongside being essential for initiating transcriptional responses associated with thermomorphogenesis [14]. Overexpression of apple MdHSF4, MdHSFB2A, and MdHSFA1D imparts heat tolerance and rescues the heat-sensitive phenotype of MdWRKY75-Ri3 [15]. Grape VdHSFA2 and VvHSFA2 are positive regulators in grape thermotolerance; meanwhile, they induce a thermotolerance response by targeting the MBF1c promoter [16]. These findings highlight the significant role of HSFs in response to environmental stresses. However, little is known about the presence or potential functions of HSFs within Eriobotrya japonica.
In this study, a bioinformatics analysis was conducted to identify and characterize members of the loquat’s HSF gene family, discovering 30 genes. Furthermore, an extensive expression analysis of these genes was performed at various growth stages and under heat-stress conditions. The findings present a preliminary analysis of HSFs in loquat and demonstrate their roles in response to high-temperature stress resistance and their potential application in genetic breeding.

2. Materials and Methods

2.1. Identification of HSF Genes in Eriobotrya japonica

The genomic data of Eriobotrya japonica cv Jiefangzhong (CNP0001531), formerly released by Su et al. (2021) [1], were obtained from the China National GeneBank database (https://db.cngb.org/cnsa, accessed on 14 September 2023). HSF protein domain data (Pfam accession number: PF00447.20) were obtained from the protein families database (http://pfam.xfam.org/, accessed on 14 September 2023) [17]. The HMM profile was constructed for the known HSF using the “hmmsearch” function in HMMER-3.3.2 software. All identified HSFs with e-values less than or equal to 1 × 10−5 were considered for downstream analysis [18]. ProtParam (https://web.expasy.org/protparam/, accessed on 15 September 2023) was used to predict the theoretical molecular weights (MWs) and isoelectric point (pI) values of these proteins [19]. The CELLO2GO program (http://cello.life.nctu.edu.tw/cello2go/, accessed on 15 September 2023) was used to predict the subcellular localization of the EjHSF genes [20].

2.2. Phylogenetic Tree Construction and Gene Structure Analysis

The HSF protein sequences of Eriobotrya japonica, Arabidopsis thaliana, Oryza sativa, Malus domestica, Prunus persica, and Gillenia trifoliata [18,21,22,23,24] were used for multiple sequence alignment using MEGA X software. The phylogenetic tree was constructed with the NJ method (1000-replicates bootstrap). MEME suite (https://meme-suite.org/meme/, accessed on 16 September 2023) was used to identify the motifs with e-values of less than 1 × 10−20 and number the 10–50 amino acids according to the corresponding e-values [25]. The gene structure of the EjHSFs was analyzed using genome annotation files.

2.3. Cis-Element Analysis

The EjHSFs upstream of the 2000 bp sequence was extracted to predict the cis-acting elements using PlantCARE database (http://bioinformatics.psb.ugent.be/webtools/plantcare/html/, accessed on 16 September 2023) [26].

2.4. Gene Duplication Events

Circos 0.69-9 software was used to analyze gene duplication events in EjHSFs via the MCScanX function [27]. The HSF gene annotation files and whole-genome sequences of Eriobotrya japonica, Arabidopsis thaliana, Oryza sativa, Malus domestica, Prunus persica, and Gillenia trifoliata were downloaded from the Joint Genome Institute database (https://genome.jgi.doe.gov/portal/, accessed on 17 September 2023). Synteny analysis of the EjHSFs and the other five species of HSF genes was performed using the MCScanX function in Circos 0.69-9 software.

2.5. Expression of EjHSF Genes

Trim Galore v0.6.10 software was used to prune the transcriptome reads, and the reads were then used for alignment with the Eriobotrya japonica genome using Hisat2 [3]. The count of each gene’s reads was calculated using StringTie v2.1.5 software and converted into FPKM to evaluate the gene expression. The FPKM values of the EjHSF genes were transformed via log2, which was visualized by constructing heat maps using TBtools-ll v2.009 software [28]. The tissue-specific expression levels for thirty EjHSF genes were referenced from Su et al.’s study (2021) [1]. This included the gene expression in different tissues (root, stem, young leaf, mature leaf, flower, inflorescence, pollen, young fruit, mature fruit, and seed tissues) of cultivated ‘Jiefangzhong’. The heat stress expression levels for thirty EjHSF genes were referenced from Chen et al.’s study (2022) [3]. Fruits in the color-changing period of ‘Wuduiyadanben’ from 8-year-old loquat trees planted in the National Germplasm Bank of Loquat (Fuzhou, China) were exposed to 40 °C in temperature-controlling boxes (Fuzhou, China) from 10:30 a.m. to 12:00 a.m. wherein the ambient temperature fluctuated around 28 ± 2 °C, and the illumination intensity was ≥1100 μmol·m−2·s−1 on 7 April 2016. Fruits with 0, 0.5, 1, and 1.5 h heat treatment were collected for physiological and molecular analyses. Peel tissues were separated immediately after the fruits were collected, frozen in liquid nitrogen, and stored at −80 °C for further analysis [3].

3. Results

3.1. Identification and Chromosomal Distribution of EjHSFs

Thirty EjHSFs were identified based on the Eriobotrya japonica genome (PF00447.20). As indicated in Table 1 and Figure 1, these genes were unevenly distributed across the 16 chromosomes of the reference genome of ‘Jiefangzhong’ loquat, except for EjHSF-A4d, which was not located on any chromosome. Chromosome 14 harbored the highest number of EjHSF genes (five), while chromosomes 1, 3, 7, 9, 10, 11, 13, and 17 contained a single copy of an EjHSF gene. In addition, three EjHSFs were found on both chromosomes 2 and 5, and two genes were identified on each of chromosomes 4, 8, 12, 15, and 16. Sixteen EjHSFs were localized on the upper arm of the chromosome, while thirteen members resided on the lower arm.
Out of the 30 members analyzed, 18 (60%), 10 (33.3%), and 2 (6.7%) belonged to the HSP-A, HSP-B, and HSP-C families, respectively. EjHSF-A1a exhibited the longest protein length of 659 aa while EjHSF-B5a exhibited the shortest of 190 aa. The MWs of these proteins ranged from 21.7 kDa (EjHSF-B5a) to 73.5 kDa (EjHSF-A3a), and their pI values ranged from 4.69 (EjHSF-B2a) to 8.86 (EjHSF-B5a). Based on the subcellular location prediction results, twenty-eight EjHSF members were predicted to be localized in the nucleus, and EjHSF-A3a and EjHSF-A7a were predicted to be localized in the cytoplasmic compartment.

3.2. Synteny Analysis

Gene duplication is a natural process that generates new genes with useful functions, promoting species development [18]. The loquat genome’s homology blocks are represented by gray lines in Figure 2. The red lines between chromosomes denote that twenty-six EjHSFs had undergone duplication events, while only three genes (EjHSF-A5a, EjHSF-B5a, and EjHSF-A4c) did not experience duplication events. The syntenic genes were located on different chromosomes from their partner, except EjHSF-A9a and EjHSF-A2a, which were located on the same chromosome (Chr-14).
A synteny analysis was performed to understand the phylogeny of the EjHSF gene family, comparing it with HSF genes from Eriobotrya japonica (30), Arabidopsis thaliana (25), Oryza sativa (25), Malus domestica (47), Prunus persica (45), and Gillenia trifoliata (34) species (Figure 3). The analysis revealed that twenty-nine EjHSFs were in sync with Malus domestica (both from the apple tribe of Rosaceae) while diverging with Prunus persica and Gillenia trifoliata (other Rosaceae species). In addition, twenty-three EjHSF genes were in sync with Vitis vinifera genes and twenty-one with Arabidopsis thaliana genes. Moreover, seventeen and fifteen EjHSF genes were in sync with Ananas comosus and Oryza sativa genes, respectively (Supplementary Table S1). Several EjHSFs were associated with multiple HSF genes in Malus domestica; for example, EjHSF-A6a was associated with four HSFs in Malus domestica.

3.3. Phylogenetic Classification of EjHSFs

The classification and evolutionary relationships of the EjHSF gene family in loquat were elucidated. Furthermore, the phylogenetic tree was constructed for the 206 HSF protein sequences, including Eriobotrya japonica (30), Arabidopsis thaliana (25), Oryza sativa (25), Malus domestica (47), Prunus persica (45), and Gillenia trifoliate (34) (Figure 4). The thirty members of the EjHSF gene family were clustered into three categories based on the clustering bootstrap value: classes A, B, and C. Among these, 18 members belonged to subgroup A, which was further divided into nine subclusters (A1, A2, A3, A4, A5, A6, A7, A8, and A9), and 10 members belonged to subgroup B, which was classified into five subclasses (B1, B2, B3, B4, and B5). Two members (EjHSF-C1a and EjHSF-C1b) belonged to C1 subclusters. Except for EjHSF-C2 subfamilies, every other EjHSF subfamily contained 1-4 EjHSF genes. Twenty-nine members of the EjHSF family had high homology with Malus domestica and Gillenia trifoliate. For example, EjHSF-A2a and EjHSF-A2b had high homology (99%) with MdHSFA2b and MdHSFA2a, respectively; EjHSF-B2c had high homology (93%) with MdHSFB2a; and EjHSF-B1a had high homology (92%) with Gtr09g3859. Proteins within each class typically exhibited similar biological functions, enabling valuable predictions regarding the future biological functions of additional members within the EjHSF gene family.

3.4. Conserved Motifs and Gene Structure Analysis of EjHSFs

The structural diversity of the EjHSFs was evaluated by analyzing all their motifs using MEME Suite. Ten distinct motifs were identified in the EjHSF proteins (motifs 1–10). Motifs 1, 2, 4, and 5 were consistently found together, suggesting a close association with SQUAMOSA promoter binding-like (SPL) proteins. Members of the same subfamily of EjHSFs exhibited similar motif compositions. For example, subfamily B contained motifs 1, 2, 4, 5, and 7; subfamily C contained motifs 1, 2, 3, 4, and 5; and subfamily A exhibited nine motifs (motifs 1–6 and 8–10). Additionally, several motifs were located at specific positions. For example, motif 5 was consistently located at the beginning of the motifs, except for EjHSF-A1a and different motifs were positioned at the end. Motif 2 always appeared between motifs 5 and 1 and motif 4 followed after motif 1 (Figure 5A,B; Supplementary Table S2).
All EjHSFs contained HSF-DNA-binding sites, and EjHSF-A1a, EjHSF-A4a, EjHSF-A4b, and EjHSF-A7a contained methltransf-12, Tup-N, TolA binding tri, and macoilin, respectively (Figure 5C). The structural and taxonomic diversity of the HSF genes in loquat was explored by comparing the genomic DNA sequences. Differences in the exon–intron structures revealed that thirty EjHSF genes had varying numbers of exons, ranging from two to six. Most EjHSF genes (22 (73.3%)) contained two exons. EjHSF-A3a exhibited the highest number of six exons and five introns. Furthermore, EjHSF-A2a and EjHSF-A2b were classified into the A2 subfamily with identical exon and intron structures consisting of four exons and three introns (Figure 5D). Generally, EjHSFs belonging to the same subfamily presented similar gene structures and potential functional diversification among its members.

3.5. Analysis of cis-Acting Elements in EjHSF Promoters

The promoter regions of EjHSFs were analyzed to gain insights into their tissue-specific expression patterns and stress responses. This study retrieved 2000 upstream sequences (the upstream regulatory region) for all thirty HSFs from the loquat genome using the gene coordinates. Upon searching against the PlantCARE databases, this study obtained 33 cis-acting regulatory elements (CAREs) that were functionally relevant and connected to the expression of HSFs (Figure 6). The promoter CAREs were categorized into three groups: abiotic and biotic stresses, phytohormone responsiveness, and plant growth and development-related elements. Most individual EjHSF genes in loquat encompassed various phytohormone response elements, such as abscisic acid (ABA) response elements (ABRE), SA hormone response elements (TCA-element), and MeJA hormone response elements (CGTCA-motif and TGACG-motif). Additionally, CAREs associated with low temperature (LTR), drought (MYC, MBS, as-1), anaerobic conditions (ARE), and other abiotic and biotic stress-related defenses were found in most of the EjHSF genes. The cis-element number of the EjHSF-A8b promoter reached 78. Among these, the number of light elements (G-box) and ABA response elements (ABRE) were 12 and 10, respectively.
The EjHSF promoters contained more than one CARE, except for the EjHSF-B4b promoter, which did not contain an element. Twenty-eight EjHSF promoters contained drought elements (MYC) and light elements (G-box). The EjHSF-B4a promoter contained only one G-box element. Twenty-seven EjHSF promoters contained drought elements (MYB). STRE elements, activated by heat shock, were found in twenty-three EjHSF promoters, indicating that these genes may regulate HSFs. The cis-element number of the EjHSF-A7b promoter reached nine. Twenty-five EjHSF promoters contained MeJA response elements (CGTCA-motif and TGACG-motif), and twenty-four promoters contained ABA response elements (ABRE) and drought elements (as-1). These findings suggest that specific cis-acting elements may regulate gene expression in different tissues, such as seeds and meristematic tissues. Furthermore, it is speculated that EjHSF genes might participate in plant growth developmental processes and respond to various abiotic and biotic stresses.

3.6. Expression Patterns of EjHSFs in Various Tissues

Further analysis was conducted using the available RNA-seq data from ten tissue types of loquat to investigate the specific expression patterns of the thirty EjHSF genes (Figure 7; Supplementary Table S3). Among them, eleven EjHSF genes were highly expressed in different tissues. For instance, EjHSF-B1a revealed high expression levels in stem, root, flower, inflorescence, young fruit, and mature fruit tissues, especially in mature fruit and stem tissues. EjHSF-A4c were highly expressed genes in stem, root, young leaf, mature leaf, young fruit, and mature fruit tissues. EjHSF-A8b showed highly expressed genes in stem, root, flower, and young fruit tissues, while EjHSF-A2a was highly expressed in seed, flower, and stem tissues. EjHSF-B2b exhibited significantly higher expression levels in root, young leaf, flower, pollen, young fruit, and mature fruit tissues, especially young fruit. Meanwhile, EjHSF-B4b was highly expressed in root and inflorescence tissues. EjHSF-C1b was highly expressed in stem and root tissues. Conversely, EjHSF-A4a was the highest expressed in roots. The EjHSF-A4d, EjHSF-B4a, and EjHSF-C1a genes indicated high transcriptional abundance in roots but exhibited low expression levels in other tissues, suggesting potential developmental roles. Meanwhile, five EjHSFs (A7b, A3b, A2b, B5a, and A6a) exhibited low expression levels in all tested tissues. It was suggested that these genes may be highly expressed in other tissues.

3.7. Expression Profiles of EjHSF Genes Under Heat Stress

This study collected and analyzed the expression data under high-temperature stresses to investigate the expression profiles of EjHSF genes under heat-stress conditions. Among the different heat stress durations applied (Figure 8; Supplementary Table S4), eleven EjHSFs (A3a, A5a, A2a, A2b, A7a, B2c, A7b, B1b, B2a, B2b, and A8b) were upregulated to different degrees, while four EjHSFs (B1a, A1b, A4d, and A8a) were downregulated. Both EjHSF-A2a and EjHSF-A2b were upregulated by up to 300 times compared with the control. Meanwhile, four EjHSFs (B2c, B2b, A7a, and A7b) were continuously increased (log2-based fold change >1) based on the processing time under heat-stress conditions. EjHSF-A3a and EjHSF-A5a initially exhibited higher expression, followed by decreased expression 1 h after the initiation of treatment. EjHSF-B1a was significantly downregulated under heat stress, whereas fourteen EjHSFs (A1a, B3a, B3b, A3b, C1a, A9a, B4a, C1b, A6a, B4b, A9b, B5a, A4a, and A4b) consistently displayed low expression levels regardless of the presence of heat stress. Meanwhile, EjHSF-A4c consistently displayed an increasing trend over time regardless of whether heat stress was present.

4. Discussion

With the publication of the loquat genome sequence, a series of HSF families have undergone genome-wide identification in the last years, such as rye (Secale cereale L.) [29], peanut (Arachis hypogaea L.) [30], peach (Prunus persica) [31], and pineapple (Ananas comosus) [32]. The loquat is an important economic fruit crop and medicinal plant. However, the yield and quality of loquat fruits are significantly influenced by various environmental stresses. Thus, characterizing gene families in the loquat genome is crucial for understanding their role in the plant’s response to environmental stresses. In this study, thirty EjHSF genes were identified in the Eriobotrya japonica genomic sequence, including eighteen EjsHSF-A, ten EjHSF-B, and two EjHSF-C genes, which reflected a similar distribution proportion to that seen among HSF genes found in other higher plants’ genomes.
EjHSF genes revealed an uneven distribution among chromosomes, suggesting genetic divergence during evolution. Twenty-nine EjHSF genes were distributed across sixteen chromosomes, with most members located on Chr-14, while no EjHSF gene was found on Chr-6. A similar phenomenon was observed in peaches, where eighteen PpHSFs were distributed among chromosomes 1–8, except Chr-6 [31]. Similarly, pineapples had nineteen AcHSF genes mapped onto its eleven chromosomes, while three AcHSF genes were located on a scaffold instead of specific chromosomes [32]. Soybeans exhibited a distribution of thirty-eight HSF genes across fifteen chromosomes, excluding chromosomes 2, 6, 7, 12, and 18 [33]. This observation reveals that some Rosaceae plant genes are not located on Chr-6.
Phylogenetic analysis divided the identified EjHSF genes into 15 families comprising 30 members. Gene families are genes from a common ancestor, often retaining similarities [34]. EjHSF-A4c and EjHSF-A4d demonstrated closer similarity to LlHSFA4 from Lilium longiflorum plant species, which positively regulates the response against B. cinerea infection via the LlWRKY33-LIHSFA4-L1CAT2 regulatory module [35]. Consequently, EjHSF-A4c and EjHSF-A4d may also participate in the disease response. Similarly, EjHSF-A1a and EjHSF-A1b belonged to the same subfamily as Solanum lycopersicum SlHSFA1a. SlHsfA1a maintains pollen thermotolerance and cellular homeostasis by enhancing antioxidant capacity and protein repair and degradation, ultimately improving pollen viability and fertility [36]. Hence, further investigation is needed to explore the potential involvement of EjHSF-A1a and EjHSF-A1b in pollen development. Moreover, EjHSF-C1a and EjHSF-C1b shared the same cluster as Pyrus betulifolia’s PbHSFC1a, known for its ability to improve drought tolerance by coordinating ABA biosynthesis and H2O2 signaling pathways [15]. This suggests that EjHSF-C1a and EjHSF-C1b might participate in drought tolerance.
Gene duplication is essential for evolution because it facilitates the production of new genes and gene functions. Twenty-six EjHSF genes had undergone duplication events. Similar results were found in chickpeas and peas [18]. Synteny analysis is a method used to compare the arrangement of genes and their relative positions on the chromosomes of different species. Synteny analysis revealed homology between the loquat HSF family and other plants belonging to the Rosaceae family, such as Malus domestica, Prunus persica, and Gillenia trifoliata. High homology was observed between EjHSFs and Malus domestica (97%), indicating the conservation of HSF gene expression during plant evolution in these two species, and these processes are likely regulated via similar mechanisms in both species [18]. However, significant divergence was noted when loquat HSF genes were compared with species such as Arabidopsis thaliana (86%), Ananas comosus (74%), and Oryza sativa (65%). Kanwar et al. [18] found that many pea PsHSF genes were conserved between peas and Arabidopsis, and eleven PsHSFs exhibited synteny with AtHSFs, providing evidence that duplication events played a crucial role in genome expansion. This study discovered that twenty-nine EjHSFs showed synteny with thirty-one apple MdHSFs, suggesting that EjHSFs might have similar functions to the apple genes.
Most EjHSF genes examined in the gene structure analysis contained two exons and one intron, suggesting their contribution to transcriptional regulation under stress conditions. EjHSF-A3a was an exception, possessing six exons and five introns, implying the possible occurrence of gene fragment insertions or deletions during the evolutionary process. Furthermore, EjHSF genes within the same subfamily, such as EjHSF-A2a and EjHSF-A2b, exhibited similar gene structures, supporting their phylogenetic relationships. Subfamily A indicated greater structural differences in the number of introns, which play an important role in gene transcriptional regulation under stress conditions. In loquats, four protein motifs (motifs 1, 2, 4, and 5) were commonly found in the EjHSF gene structure, indicating their close association with SPL proteins. Motif 7 was exclusively involved in class B, while motif 3 was specially contained in classes A and C, but not B. These results suggest that genes from the same subfamily tend to cluster together based on similar genetic compositions and structures to those observed in other plants. In alfalfa, protein motifs 1 and 3 were highly conserved across all MsHSP genes, while motifs 7 and 9 were only present in HSF A. Motif 8 was exclusive to HSF class B [37]. Similarly, peaches exhibited motifs 1–3 present in each PpHSF, while motif 4 occurred only in classes A and B [31]. Pineapples’ classes A and B mostly contained motif 4. In contrast, motif 3 existed solely in class A and C HSFs, not class B [32]. Conserved protein motifs constitute the structural basis for the biological function of HSFs. The same protein is distributed in different HSF subfamilies among different plants.
In peaches, every promoter contained at least two MYB elements. The next most common elements were MYC, CGTCA- and TGACG-motifs, ARE, ERE, and MBS [31]. The STRE motif was the most common element in petunias, and the other cis-acting elements were ARE and Box 4. The PtHSF01 promoter in kiwifruits contains the highest number of cis-acting elements (class A), while the PtHSF23 (class C) promoter had the fewest cis-acting elements [38]. In rye, the ScHSF gene covers many phytohormone response elements, including ABR and MeJA hormone response elements (CGTCA-motif and TGACG-motif) [29]. In asparaguses, almost all AoHSF members were found to possess both hormone- and stress-related features, with AoHSF06 identified as containing an RY element related to sprouting [39]. Overall, HSF promoters in different plants universally express hormone response- and stress-related elements. The analysis of cis-acting elements within the EjHSF promoters revealed thirty-three CAREs that were functionally relevant and connected to the expression of HSFs. Twenty-eight EjHSF promoters contained drought elements (MYC) and light elements (G-box). Twenty-seven EjHSF promoters contained drought elements (MYB). Twenty-five EjHSF promoters contained MeJA response elements (CGTCA-motif and TGACG-motif), and twenty-four promoters contained ABA response elements (ABRE) and drought elements (as-1). STRE elements, activated by heat shock, were found in twenty-three EjHSF promoters, indicating that these genes may regulate HSFs. EjHSF genes may also be involved in multiple signaling pathways and play a critical role in regulating development, abiotic stress, and hormone synthesis in loquats.
In the tomato, SlHSFA1a plays a crucial role in maintaining pollen heat resistance and improving pollen viability and fertility [36]. In the loquat, the expression patterns of EjHSFs in different tissues and organs were analyzed, and distinct tissue-specific expression patterns were revealed among different EjHSFs. Eleven EjHSFs (A2a, A4a, A4c, A4d, A8b, B1a, B2b, B4a, B4b, C1a, and C1b) were highly expressed in different tissues. Ten EjHSFs (A4a, A4c, A4d, A8b, B1a, B2b, B4a, B4b, C1a, and C1b) had high expression levels in roots. Among them, EjHSF-A4a showed the highest expression. EjHSF-C subfamilies exhibited high expression levels in roots but low levels in fruits, indicating their potential role in plant growth regulation. EjHSF-B1a exhibited the highest expression levels in mature fruits and stems, while EjHSF-B2b showed the highest expression levels in young fruits. Conversely, five EjHSFs (A7b, A3b, A2b, B5a, and A6a) revealed low expression across all tissues examined. Further investigation is needed to explore the potential function of EjHSFs.
Most plant HSFs known to date play crucial regulatory roles in response to environmental stress, particularly heat stress. For example, CaHSFA2 was activated by HSFA6a, which was directly targeted and activated by CaHDZ15 and further improved the heat resistance of peppers [40]. Grape VdHSFA2 of the heat-tolerant Vitis davidii ‘Tangwei’ and VvHSFA2 of the heat-sensitive Vitis vinifera ‘Jingxiu’ are positive regulators in grape thermotolerance, and VdHSFA2 can confer higher heat resistance than VvHSFA2 [16]. Overexpression of lily LlHsfA2, rice OsHsfA2e, and tall fescue FaHsfA2c enhanced plant heat resistance [41,42,43]. In the research results, EjHSF-A2a and EjHSF-A2b belonged to the HSF-A2 subfamily. The phylogenetic analysis showed that EjHSF-A2a and EjHSF-A2b were highly homologous to AtHSFA2a. EjHSF-A2a was highly expressed in seed, flower, and stem tissues, while EjHSF-A2b exhibited low expression levels in all tested tissues. The transcriptional abundance of EjHSF-A2a and EjHSF-A2b changed by over 300 times after 1 h of heat stress treatment, wherein the EjHSF-A2a expression level was notably upregulated. Their specific functions have been followed up for further study.
TrHSFB2a of white clover (Trifolium repens) was highly homologous to MtHSFB2b, CarHSFB2a, AtHSFB2b, and AtHSFB2a and exhibited more strongly induced high expression after heat stress treatment for 3 h and then reduced continuously. Under drought-, salt-, and heat-stress conditions, the overexpression of TrHSFB2a in Arabidopsis significantly reduced drought, salt, and heat resistance. Meanwhile, silencing AtHSFB2a via RNA interference in Arabidopsis showed an opposite trend to wild-type Arabidopsis plants [12]. These observations were also observed in the transgenic lines developed via the overexpression and RNA interference homolog of OsHSFB2b in rice [44] and the overexpression of chickpea CarHSFB2b in Arabidopsis [45]. Apple plants that overexpressed MdHSF4, MdHSFB2a, and MdHSFA1d exhibited improved heat resistance and rescued the heat-sensitive phenotype of MdWRKY75-Ri3 [15]. In this study, EjHSFs-B2 was highly homologous to MdHSFB2 and AtHSFB2. EjHSF-B2b exhibited significantly higher expression levels in young fruits and showed high expression in root, young leaf, flower, pollen, and mature fruit tissues. Meanwhile, the expressions of EjHSF-B2b, EjHSF-B2c, and EjHSF-B2b continuously increased with the processing time under heat-stress conditions, suggesting their potentially similar functions.
In the tomato, SlHSFB1 suppression strongly enhanced the induction of HSP due to the higher activity of other HS-induced HSFs under heat stress (HS), resulting in increased heat resistance compared with the wild type. SlHSFB1 overexpression stimulates the co-activator function of SlHSFB1 and, consequently, the accumulation of HS-related proteins under non-stress conditions. Plants with enhanced SlHSFB1 levels show high heat resistance [46]. Fragkostefanakis et al. [46] indicated that SlHSFB1 acts as both a co-activator of HSFA1a and a transcriptional repressor of other HSFs (HSFA1b and HSFA2). This dual function explains the activation of chaperones to enhance protection and regulate the balance between growth and stress responses upon deviations from the homeostatic levels of SlHSFB1 in tomatoes [46]. In this study, EjHSF-B1a belonged to the same EjHSF-B1 subclusters. EjHSF-B1a revealed the highest expression levels in mature fruits and stems and high expression levels in other tissues (root, flower, inflorescence, and young fruit tissues). EjHSF-B1a was significantly downregulated in loquat fruits under heat stress, while EjHSF-B1b expression slightly increased. Whether EjHSFB1 has the same function as SlHSFB1 requires further study.

5. Conclusions

In conclusion, this study showed that the diverse expression patterns of EjHSFs collectively contribute to the loquat’s adaptation to heat-stress conditions, highlighting the important regulatory role of HSF genes during Eriobotrya japonica’s response to heat stress. This warrants further investigation and attention for genomic improvement and medical applications related to high-temperature resistance.

Supplementary Materials

The following supporting information can be downloaded at: https://www.mdpi.com/article/10.3390/horticulturae10111195/s1, Table S1: Synteny analysis; Table S2: Motif consensus; Table S3: The expression profile of EjHSFs in different tissues; Table S4: The expression profile of EjHSFs under heat stress [1,3].

Author Contributions

C.D. and J.J. conceived and designed the experiments and obtained the funding. C.D., Y.C., X.C. and W.W. performed the experiments and analyzed the data. C.D. and Y.C. drafted the manuscript. All authors have read and agreed to the published version of the manuscript.

Funding

This work is supported by the Collaborative Innovation Project from the People’s Government of Fujian Province & Chinese Academy of Agricultural Sciences (XTCXGC2021006); the Technology Innovation Team Program (CXTD2021004) as well as the Subject Promotion Project for Logan and Loquat Research from Fujian Academy of Agricultural Science and the Natural Science Foundation of Fujian Province (2023R1085).

Data Availability Statement

Data are available upon request from the corresponding author due to funders’ legal restrictions and requirements.

Conflicts of Interest

The authors declare no conflicts of interest. The funders had no role in the design of the study, in the collection, analyses or interpretation of data, in the writing of the manuscript, or in the decision to publish the results.

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Figure 1. The localization of HSF family genes on loquat chromosomes. The sale represents the megabases (Mbs), the numerical value represents the chromosome number (LG for linkage group), and the chromosomes’ differences in color represent the gene density, the color ranges from white to blue to red, the higher the density.
Figure 1. The localization of HSF family genes on loquat chromosomes. The sale represents the megabases (Mbs), the numerical value represents the chromosome number (LG for linkage group), and the chromosomes’ differences in color represent the gene density, the color ranges from white to blue to red, the higher the density.
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Figure 2. Chromosomal distribution and duplication events. The 29 putative segmental duplicated pairs of the EjHSF genes were investigated using MCScanX and linked by the colored lines, respectively. The gray line indicates all the putative segment duplication pairs in the loquat genome while the red line links the EjHSF segment duplication pairs.
Figure 2. Chromosomal distribution and duplication events. The 29 putative segmental duplicated pairs of the EjHSF genes were investigated using MCScanX and linked by the colored lines, respectively. The gray line indicates all the putative segment duplication pairs in the loquat genome while the red line links the EjHSF segment duplication pairs.
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Figure 3. Synteny analysis of HSF genes between loquat and five other plant species. The gray line in the background represents the collinear blocks between Eriobotrya japonica and six other plant species (Arabidopsis thaliana, Oryza sativa, Vitis vinifera, Malus domestica, Prunus persica, and Gillenia trifoliata) while the blue line exhibits the syntenic HSF gene pairs.
Figure 3. Synteny analysis of HSF genes between loquat and five other plant species. The gray line in the background represents the collinear blocks between Eriobotrya japonica and six other plant species (Arabidopsis thaliana, Oryza sativa, Vitis vinifera, Malus domestica, Prunus persica, and Gillenia trifoliata) while the blue line exhibits the syntenic HSF gene pairs.
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Figure 4. Protein-based phylogenetic tree of HSF gene family. ▲, ■, ★, ●, ●, and ● represent the phylogenetic analysis of HSFs in Eriobotrya japonica (Ej), Arabidopsis thaliana (At), Oryza sativa (Os), Malus domestica (Md), Prunus persica (Pp), and Gillenia trifoliata (Gt), respectively. The other genes mentioned in the chart are Lilium longiflorum (Ll), Solanum lycopersicum (Sl), and Pyrus betulifolia (Pb).
Figure 4. Protein-based phylogenetic tree of HSF gene family. ▲, ■, ★, ●, ●, and ● represent the phylogenetic analysis of HSFs in Eriobotrya japonica (Ej), Arabidopsis thaliana (At), Oryza sativa (Os), Malus domestica (Md), Prunus persica (Pp), and Gillenia trifoliata (Gt), respectively. The other genes mentioned in the chart are Lilium longiflorum (Ll), Solanum lycopersicum (Sl), and Pyrus betulifolia (Pb).
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Figure 5. The motif and gene structure organizations of EjHSF members. (A) The evolutionary tree of loquat HSF family members. (B) Protein motif locations of loquat HSF members. The protein sequences were subjected to Motif Search based on homology. Boxes represent motifs, and different colors represent different motifs. (C) Conserved binding sites of EjHSF genes. Different colored boxes represent different types of conserved binding sites. (D) Gene structures of EjHSF genes. Intron–exon size distribution of HSF family genes. The nucleotide sequences of these genes were used for analysis. The red box represents exons, and the line represents introns.
Figure 5. The motif and gene structure organizations of EjHSF members. (A) The evolutionary tree of loquat HSF family members. (B) Protein motif locations of loquat HSF members. The protein sequences were subjected to Motif Search based on homology. Boxes represent motifs, and different colors represent different motifs. (C) Conserved binding sites of EjHSF genes. Different colored boxes represent different types of conserved binding sites. (D) Gene structures of EjHSF genes. Intron–exon size distribution of HSF family genes. The nucleotide sequences of these genes were used for analysis. The red box represents exons, and the line represents introns.
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Figure 6. Distribution of cis-acting elements in promoters of EjHSF gene family members. The cis-element number of each EjHSF gene promoter shows in each box with green color, the greener the color, the greater the number of cis-element.
Figure 6. Distribution of cis-acting elements in promoters of EjHSF gene family members. The cis-element number of each EjHSF gene promoter shows in each box with green color, the greener the color, the greater the number of cis-element.
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Figure 7. Expression heatmap of EjHSF genes in different tissues (root, stem, young leaf, mature leaf, flower, inflorescence, pollen, young fruit, mature fruit, and seed tissues). FPKM values of EjHSF genes were transformed via log2.
Figure 7. Expression heatmap of EjHSF genes in different tissues (root, stem, young leaf, mature leaf, flower, inflorescence, pollen, young fruit, mature fruit, and seed tissues). FPKM values of EjHSF genes were transformed via log2.
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Figure 8. Heatmap revealing differential expression patterns of EjHSF genes in the color-changing period of ‘Wuduiyadanben’ loquat fruit under control conditions and 40 °C heat treatment. 0 h: control group, 0.5 h: 0.5 h under heat stress, 1 h: 1 h under heat stress, and 1.5 h: 1.5 h under heat stress. The number in the box represents the raw TPM (transcripts per kilobase of exon model per million mapped reads). The different colors represent FPKM-normalized log2-converted counts, where red represents high expression, and green represents low expression.
Figure 8. Heatmap revealing differential expression patterns of EjHSF genes in the color-changing period of ‘Wuduiyadanben’ loquat fruit under control conditions and 40 °C heat treatment. 0 h: control group, 0.5 h: 0.5 h under heat stress, 1 h: 1 h under heat stress, and 1.5 h: 1.5 h under heat stress. The number in the box represents the raw TPM (transcripts per kilobase of exon model per million mapped reads). The different colors represent FPKM-normalized log2-converted counts, where red represents high expression, and green represents low expression.
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Table 1. Summary of heat shock transcription factors (EjHSFs) in Eriobotrya japonica.
Table 1. Summary of heat shock transcription factors (EjHSFs) in Eriobotrya japonica.
GeneIDGeneNameChrStartEndpIMw/kDaProtein Length/aaSubcellular Location
Ej00038938EjHSF-A1achr0515,417,69115,423,3715.5373,313.98659Nuclear
Ej00005590EjHSF-A1bchr105,669,4095,673,3975.1259,636.99534Nuclear
Ej00081356EjHSF-A2achr1439,162,49339,164,5145.3644,862.03402Nuclear
Ej00011860EjHSF-A2bchr086,128,6976,130,5314.8545,921.50411Nuclear
Ej00044128EjHSF-A3achr1546,335,17546,337,6965.0573,528.70654Cytoplasmic
Ej00008404EjHSF-A3bchr1240,198,21240,200,7715.0161,348.73553Nuclear
Ej00050337EjHSF-A4achr09837,765841,4205.5049,328.06434Nuclear
Ej00001403EjHSF-A4bchr179,213,3889,217,2905.2349,924.67440Nuclear
Ej00028977EjHSF-A4cchr057,022,2967,024,7145.2347,357.55420Nuclear
Ej00000248EjHSF-A4dutg0_pilon3,628,9173,630,8685.8647,689.09423Nuclear
Ej00014341EjHSF-A5achr1139,174,04039,177,4975.2953,735.29485Nuclear
Ej00040219EjHSF-A6achr1618,736,54518,738,1524.8540,867.55355Nuclear
Ej00083177EjHSF-A7achr0336,894,50036,896,9755.7047,676.50418Cytoplasmic
Ej00044555EjHSF-A7bchr046,817,9116,820,2455.9445,551.86400Nuclear
Ej00062710EjHSF-A8achr131,248,5071,251,3134.9046,788.41412Nuclear
Ej00056238EjHSF-A8bchr161,718,7981,721,5394.7647,005.87414Nuclear
Ej00045930EjHSF-A9achr1425,583,99325,586,5054.8753,371.73479Nuclear
Ej00017485EjHSF-A9bchr02313,403315,3105.2254,382.91484Nuclear
Ej00027446EjHSF-B1achr1530,589,33130,592,2346.3532,374.30294Nuclear
Ej00019640EjHSF-B1bchr0215,089,35715,105,6685.4836,813.31331Nuclear
Ej00094962EjHSF-B2achr0429,569,08129,570,6214.6936,024.64334Nuclear
Ej00006997EjHSF-B2bchr0736,221,46236,222,8566.3934,474.80307Nuclear
Ej00050877EjHSF-B2cchr0134,673,16534,674,5948.8433,814.23297Nuclear
Ej00023119EjHSF-B3achr147,081,2227,084,7947.5627,874.44243Nuclear
Ej00004040EjHSF-B3bchr1230,632,18330,636,3378.4627,793.39243Nuclear
Ej00081580EjHSF-B4achr1437,493,34537,495,0297.8143,447.70384Nuclear
Ej00045578EjHSF-B4bchr089,403,0469,404,7107.1343,143.32381Nuclear
Ej00066400EjHSF-B5achr055,238,6265,240,8938.8621,733.83190Nuclear
Ej00047075EjHSF-C1achr1427,775,18727,776,3525.4537,333.45334Nuclear
Ej00017302EjHSF-C1bchr022,833,3482,834,5286.1437,977.44337Nuclear
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Deng, C.; Chen, Y.; Wei, W.; Chen, X.; Jiang, J. Genome-Wide Identification of Heat Shock Transcription Factor Family and Key Members Response Analysis to Heat Stress in Loquat. Horticulturae 2024, 10, 1195. https://doi.org/10.3390/horticulturae10111195

AMA Style

Deng C, Chen Y, Wei W, Chen X, Jiang J. Genome-Wide Identification of Heat Shock Transcription Factor Family and Key Members Response Analysis to Heat Stress in Loquat. Horticulturae. 2024; 10(11):1195. https://doi.org/10.3390/horticulturae10111195

Chicago/Turabian Style

Deng, Chaojun, Yongping Chen, Weilin Wei, Xiuping Chen, and Jimou Jiang. 2024. "Genome-Wide Identification of Heat Shock Transcription Factor Family and Key Members Response Analysis to Heat Stress in Loquat" Horticulturae 10, no. 11: 1195. https://doi.org/10.3390/horticulturae10111195

APA Style

Deng, C., Chen, Y., Wei, W., Chen, X., & Jiang, J. (2024). Genome-Wide Identification of Heat Shock Transcription Factor Family and Key Members Response Analysis to Heat Stress in Loquat. Horticulturae, 10(11), 1195. https://doi.org/10.3390/horticulturae10111195

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