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Article

Genome-Wide Characterization of HSP90 Gene Family in Chinese Pumpkin (Cucurbita moschata Duch.) and Their Expression Patterns in Response to Heat and Cold Stresses

by
Yanping Hu
1,2,†,
Tingting Zhang
2,3,†,
Peng Wang
2,
Yuxin Li
2,
Min Wang
1,
Baibi Zhu
1,
Daolong Liao
1,
Tianhai Yun
1,
Wenfeng Huang
1,
Yisong Chen
1,
Wen Zhang
1,* and
Yang Zhou
2,*
1
Key Laboratory of Vegetable Biology of Hainan Province, Hainan Vegetable Breeding Engineering Technology Research Center, The Institute of Vegetables, Hainan Academy of Agricultural Sciences, Haikou 571199, China
2
Key Laboratory for Quality Regulation of Tropical Horticultural Crops of Hainan Province, School of Horticulture, Hainan University, Haikou 570228, China
3
Xiangyang Academy of Agricultural Sciences, Xiangyang 441057, China
*
Authors to whom correspondence should be addressed.
†
These authors contributed equally to this work.
Agronomy 2023, 13(2), 430; https://doi.org/10.3390/agronomy13020430
Submission received: 28 December 2022 / Revised: 19 January 2023 / Accepted: 30 January 2023 / Published: 31 January 2023
(This article belongs to the Section Horticultural and Floricultural Crops)
Browse Figures
Versions Notes

Abstract

:
Heat shock protein 90 (HSP90) plays critical roles in plant growth and development, as well as in response to abiotic stresses such as heat and cold. To comprehensively analyze the HSP90 gene family and determine the key HSP90 gene responsive to temperature stress in pumpkin (Cucurbita moschata Duch.), bioinformatics and molecular biology techniques were used in this study. A total of 10 CmoHSP90 genes were identified from the pumpkin genome, encoding amino acids of 567–865, with protein molecular weight of 64.32–97.36 kDa. Based on the phylogenetic analysis, they were classified into four groups. The members in each group contained similar conserved motifs and gene structures. The 10 CmoHSP90 genes were distributed on the 9 chromosomes of C. moschata. Four pairs of segmental duplication genes (CmoHSP90-1/CmoHSP90-10, CmoHSP90-2/CmoHSP90-7, CmoHSP90-3/CmoHSP90-6, and CmoHSP90-4/CmoHSP90-9) were detected. Synteny analysis revealed that 10 C. maxima HSP90 genes and 10 C. moschata HSP90 genes were orthologous genes with 17 syntenic relationships. Promoter analysis detected 23 cis-acting elements including development-, light-, stress-, and hormone-related elements in the promoter regions of pumpkin HSP90 genes. Further analysis showed that the transcript levels of CmoHSP90-3 and CmoHSP90-6 were remarkably up-regulated by heat stress, while CmoHSP90-6 and CmoHSP90-10 were significantly up-regulated by cold stress, suggesting that these HSP90 genes play critical roles in response to temperature stress in pumpkins. The findings will be valuable for understanding the roles of CmoHSP90s in temperature stress response and should provide a foundation for elucidating the function of CmoHSP90s in C. moschata.

1. Introduction

Global warming and climate change will increase the global mean temperature by 3–5 °C in the near future [1]. The rising temperature will pose serious challenges to plant growth and yield, resulting in a large economic loss [2]. Therefore, it is urgent to improve the heat tolerance of crops.
Plants have developed molecular mechanisms for adapting to environmental stimuli during the long course of evolution. To avoid destruction of cellular homeostasis, plants can rapidly synthesize a large amount of heat shock proteins (HSPs) in cells when subjected to temperature stress. HSPs play important roles in guiding protein transmembrane transport, promoting degradation of unstable proteins, enhancing formation and transcription of signaling molecules, and activating transcription factors [3]. According to their molecular weights, HSPs can be divided into five groups: HSP20 (sHSPs), HSP60, HSP70, HSP90, and HSP100 [4]. Heat shock protein 90 (HSP90) is a protein family with conserved structure and complex cellular functions, which can be induced to express and play critical functions when organisms are affected by environmental stimuli [5,6]. HSP90 has three distinct regions: the C-terminal, containing the dimerization region; the middle region (M), the main substrate protein binding site; and the N-terminal, containing ATP binding and hydrolysis sites [7]. The HSP90 protein plays important roles in plant seed germination, seedling growth, and other life processes [8,9], as well as in protein folding, processing, transportation, and degradation [4]. The HSP90 gene is constitutively expressed in cells, but its expression is dramatically increased severalfold under abiotic stress (mainly heat stress) [10,11]. Under heat stress, HSP90 can bind to folded and denatured proteins to maintain protein homeostasis in cells, and can activate the activity of substrate proteins, thus exerting its physiological functions. In addition, HSP90 can inhibit the function of heat shock factor (HSF) and negatively regulate the transcription of targeted genes [12].
The response of HSP90 to abiotic stress has been well studied and reported [13,14], and HSP90 genes have been found to be up-regulated under heat stress [15]. For instance, the expression levels of rice OsHSP90.1 and Arabidopsis thaliana AtHSP90 were observed to be up-regulated under heat stress [11,16]. Heat stress was found to have a stronger induction effect on tobacco HSP90s than ABA (abscisic acid), PEG (polyethylene glycol), NaCl, and cold stresses. The expression of tobacco NtHSP90-9 reached the highest level at 6 h, with an 18-fold increase compared with the initiate time [17]. In soybean, 12 GmHSP90 genes were found to be strongly induced by high-temperature treatment, and the plants transformed with these genes showed improved high-temperature and drought tolerance [18]. The soybean plants overexpressing GmHSP90A2 showed higher heat resistance by increasing chlorophyll content and decreasing malondialdehyde content when plants were incubated at 50 °C for 2 h [19], suggesting that GmHSP90A2 positively regulates the heat tolerance of soybeans [19]. Similarly, Arabidopsis AtHSP90-1 mutant displayed reduced heat tolerance [20]. Agarwal et al. [21] found that the expression levels of HSP90 genes in each tissue of heat-tolerant chickpeas were higher than those of heat-sensitive chickpeas, suggesting that HSP90s participate in the response process of heat stress. Cassava MeHSP90.9 can regulate drought resistance by recruiting MeWRKY20 and MeCatalase1 proteins [22]. However, studies showed that overexpression of HSP90 can reduce the heat tolerance of Arabidopsis. It was found that the germination rates and fresh weights of A. thaliana plants overexpressing AtHSP90.7, AtHSP90.2, and AtHSP90.5 were significantly lower than those of the wild type under drought and salt stress, suggesting that these genes can reduce drought resistance and salt tolerance of plants [23]. AtHSP90.3 overexpression reduced the heat tolerance of transgenic A. thaliana when exposed to 45 °C, indicating the critical roles of HSP90 in plant heat tolerance [24]. Taken together, these studies indicate the important roles of HSP90 family genes in plant heat tolerance.
Pumpkin is an important vegetable crop, belonging to the genus of Cucurbita and the family of Cucurbitaceae. There are three main cultivated species of pumpkin: American pumpkin (Cucurbita pepo L.), Chinese pumpkin (Cucurbita moschata Duch.), and Indian pumpkin (Cucurbita maxima Duch.) [25]. Among them, C. moschata is best adapted to hot climates and is often cultivated in tropical and subtropical regions, while C. maxima is susceptible to disease and heat stress [26]. The heat resistance of interspecific hybrids F1 C. moschata × C. pepo and C. moschata × C. maxima was remarkably enhanced when compared with that of their parents C. pepo and C. maxima, respectively, suggesting the heat tolerance genes of C. moschata can be used to improve the heat tolerance of other pumpkin varieties [27,28]. In fact, C. moschata is more frequently utilized as rootstock for improvement of cucurbit stress tolerance [29]. Studies on pumpkin HSP gene functions are quite few, and the mechanism underlying C. moschata’s response to abiotic stresses remains unclear. In one previous study, 33 Cucurbita moschata HSP20 members were identified, and several candidate CmoHSP20 genes responding to heat stress were screened [30]. In this study, for the first time, the HSP90 family was identified, and the physicochemical characteristics, chromosomal location, expression patterns, cis-elements, gene structures, and conserved domains of the genes were analyzed. This study provides information on CmoHSP90 gene functions and a reference for molecular breeding of cucurbit vegetable crops.

2. Materials and Methods

2.1. Genome-Wide Identification of the HSP90 Genes in Cucurbita moschata

The whole genome sequence was obtained from the Cucurbita moschata Genome Database (CuGenDB; http://cucurbitgenomics.org/, accessed on 11 December 2021). To identify the CmoHSP90 candidate genes, a BLASTP search was performed in the database with A. thaliana and Oryza sativa HSP90 protein sequences as queries (the threshold e-value was set to 1 × 10−5) [31]. Then, the conserved HSP90 domain (PF00183) was confirmed by submitting the output putative HSP90 protein sequences to the Conserved Domain Database (CDD; https://www.ncbi.nlm.nih.gov/Structure/bwrpsb/bwrpsb.cgi, accessed on 25 July 2022), the Simple Modular Architecture Research Tool (SMART; http://smart.embl.de/smart/batch.pl, accessed on 25 July 2022), and the Pfam (the protein family database; http://pfam.xfam.org/, accessed on 25 July 2022). After removing the redundant sequences and the predicted protein sequences lacking the common HSP90 domain, the candidate genes were assigned as C. moschata HSP90s, and 10 CmoHSP90 genes were finally obtained.

2.2. Physical and Chemical Properties and Position on Chromosomes

The information on the molecular weights, isoelectric points, and number of amino acids of CmoHSP90 was predicted using the ExPASy software (http://web.expasy.org/protparam/, accessed on 30 July 2022), and the transmembrane of the CmoHSP90 proteins was predicted using the TMHMM (http://www.cbs.dtu.dk/services/TMHMM/, accessed on 30 July 2022). The protein sequences were uploaded to PSORT (https://www.genscript.com/psort.html, accessed on 30 July 2022) for prediction of subcellular localization. The CmoHSP90 gene chromosomal positions were determined from the pumpkin genome, and MapChart software was used for mapping [32].

2.3. Construction of Phylogenetic Tree of HSP90 Proteins

The sequences of HSP90 proteins of watermelon (Citrullus lanatus), bottle gourd (Lagenaria siceraria), cucumber (Cucumis sativus), sponge gourd (Luffa cylindrica), bitter gourd (Momordica charantia), melon (Cucumis melo), wax gourd (Benincasa hispida), and Cucurbita maxima were obtained from the Cucurbit Genomic Database [31]. The HSP90 protein sequences of chayote (Sechium edule), snake gourd (Trichosanthes anguina), and monk fruit (Siraitia grosvenorii) were identified using the identification method for the pumpkin HSP90 gene family. Multiple HSP90 protein sequences obtained from pumpkin, rice, Arabidopsis, and the 11 Cucurbitaceous crops mentioned above were aligned with default parameters using the MEGA-X software, and based on the alignment results, a phylogenetic tree was then constructed using the maximum-likelihood (ML) method (the value of bootstrap was set to 1000).

2.4. Structure Analysis of HSP90 Gene in Pumpkin

The conserved motifs of CmoHSP90 proteins were identified using the Multiple Em for Motif Elicitation (MEME) program (MEME Suite 5.3.3; http://meme-suite.org/tools/meme, accessed on 31 August 2022) with the following parameters: the motif width of 6 to 100 amino acid residues and the maximum motif number of 10. The conserved HSP90 domains were obtained from the Pfam database and aligned with the DNAMAN software. The related genome sequences of CmoHSP90s were obtained using TBtools [33], and the gene exon-intron information was obtained from the CDS, followed by visualization of conserved gene structures and motifs using TBtools.

2.5. Analysis of Gene Duplication and Synteny of HSP90 Genes

Gene duplication of CmoHSP90 was analyzed with TBtools based on the pumpkin genome and its annotation file. TBtools was used to assess the nucleotide substitution parameters Ks (synonymous) and Ka (nonsynonymous), and the Ka/Ks ratio was then calculated. Synteny relationships of pumpkin with Arabidopsis and other Cucurbitaceous crops were generated using MCScanX in TBtools.

2.6. Prediction of Cis-Regulatory Elements (CREs) for Pumpkin HSP90 Gene Promoters

The CREs were identified by submitting the promoter sequence (2.0 kb upstream sequence of the start codon) of each CmoHSP90 gene extracted by TBtools to PlantCARE (http://bioinformatics.psb.ugent.be/webtools/plantcare/html/, accessed on 31 August 2022) [34]. The results including the types and numbers of CREs were visualized by TBtools.

2.7. The Network of Protein-Protein Interaction

The interactions between CmoHSP90 and other proteins were analyzed by submitting the CmoHSP90 sequences to the STRING software (https://string-db.org/, accessed on 7 September 2022) [35]. A ‘single protein by sequence’ method was applied, and ‘Arabidopsis thaliana’ was selected as the organism for analysis. The PPI networks were constructed and visualized using Cytoscape (version 3.9.1) [36].

2.8. Stress Treatments

The seeds of pumpkin cultivar Miben 2 (Cucurbita moschata cv. Miben 2) were incubated at 37 °C in the dark for 2 days to sprout and then grown under the following conditions: relative humidity of 70%, temperature of 25 °C, and a light/dark cycle of 12 h/12 h [30]. Uniform seedlings with three true leaves were incubated at 42 °C and 4 °C, respectively, for heat and cold stress. After treatment for 0, 3, 6, 9, 12, or 24 h, leaf and root samples were collected, immediately frozen in liquid nitrogen, and then stored at −80 °C for RNA extraction at a later time. Three independent replicates were included in each treatment, and for each replicate, the samples were collected from five plants.

2.9. Expression Profile Analysis of CmoHSP90 Genes by Real-Time Quantitative PCR (RT-qPCR)

RT-qPCR was performed as described previously, with CmoActin gene as an internal control [25]. Supplementary Table S1 lists all the primers used. The relative expression levels of CmoHSP90 genes were calculated using the 2−ΔΔCT method [37]. All data were calculated using the expression levels at different time points (3, 6, 9, 12, or 24 h) divided by that initial time (0 h) and are presented as the means ± standard error (SE) of three replicates. The differences were analyzed using Student’s t-test. Means for which p < 0.05 were considered statistically significantly different.

3. Results

3.1. Genome-Wide Identification and Chromosomal Location of HSP90 Family Genes in Pumpkin

The pumpkin HSP90 family genes were identified using Arabidopsis thaliana and rice HSP90 protein sequences as query sequences. The protein containing the conserved domain of HSP90 was identified as pumpkin HSP90 protein by analyzing the conserved domain of the protein in the SMART, Pfam, and CDD databases. The pumpkin HSP90 gene family contains 10 members, named CmoHSP90-1 to CmoHSP90-10 according to their positions on the chromosome (Table 1). They were 567 (CmoHSP90-1)~865 (CmoHSP90-10) amino acids (aa) long with molecular weight (MW) 64.32~97.36 kDa. The predicted isoelectric point (pI) ranged from 4.89 (CmoHSP90-2) to 8.22 (CmoHSP90-10). The transmembrane domain prediction showed that CmoHSP90-10 contained two transmembrane structures, while the other proteins contained no transmembrane domain (TMD). The subcellular localization prediction showed that CmoHSP90-1, CmoHSP90-2, and CmoHSP90-7 could localize in mitochondria, CmoHSP90-3, CmoHSP90-6, and CmoHSP90-10 could localize in endoplasmic reticulum, CmoHSP90-4 and CmoHSP90-8 possibly localized in cytoplasm and nucleus, and CmoHSP90-5 and CmoHSP90-9 could localize in cytoplasm (Table 1).

3.2. Phylogenetic Relationship of the CmoHSP90 Proteins in Pumpkin

To understand their evolutionary relationship, the HSP90 protein sequences from 14 plant species including pumpkin, C. sativus, B. hispida, L. siceraria, L. cylindrica, A. thaliana, O. sativa, C. maxima, C. melo, M. charantia, C. lanatus, S. edule, T. anguina, and S. grosvenorii (see gene IDs shown in Supplementary Table S2) were aligned, and the construction of an ML evolutionary tree was performed using MEGA-X. The phylogenetic tree indicates that the 100 HSP90 proteins were divided into four groups (Group A to Group D), among which CmoHSP90-1, CmoHSP90-2, CmoHSP90-7, and CmoHSP90-10 were clustered in Group A; CmoHSP90-3 and CmoHSP90-6 were clustered in Group B; CmoHSP90-8 was in Group C; and CmoHSP90-4, CmoHSP90-5, and CmoHSP90-9 were clustered in Group D (Figure 1). A similar distribution pattern was observed for HSP90 proteins in other species (Supplementary Figure S1).

3.3. Conserved Gene Structures and Motifs of the CmoHSP90 Genes

The conserved motif of the CmoHSP90 protein was analyzed using MEME software, and results showed that all CmoHSP90 proteins contained 10 motifs except for CmoHSP90-1 and CmoHSP90-10 (Figure 2A), and the motif length was 21~88 amino acids (Supplementary Figure S2, Supplementary Table S3). The order of the 10 motifs in the CmoHSP90 protein was motif 2-motif 6-motif 4-motif 10-motif 8-motif 7-motif 5-motif 3-motif 1-motif 9. However, CmoHSP90-1 lacked motifs 6, 7, and 1, and CmoHSP90-10 only contained motifs 2, 6, and 9. The multiple sequences of CmoHSP90 were aligned using DNAMAN software, and it was found that the CmoHSP90 protein contained ATP/ADP binding domain in the N-terminus (155 amino acids in length) and HSP90 domain in the C-terminus (501 to 550 amino acids in length), and the C-terminus was formed by full-length motifs 8, 7, 5, 1, and 9 (Figure 3).
The gene structure analysis showed that the 10 CmoHSP90 genes contained a number of introns (2~18) of varying length and position (Figure 2B). CmoHSP90-4, CmoHSP90-5, and CmoHSP90-9 contained two introns, CmoHSP90-8 contained three introns, CmoHSP90-3 and CmoHSP90-6 contained 14 introns, CmoHSP90-1 and CmoHSP90-10 contained 16 introns, and CmoHSP90-2 and CmoHSP90-7 contained 18 introns (Supplementary Table S4).

3.4. Chromosome Location and Synteny Analysis of CmoHSP90 Genes

To examine the chromosomal distributions of CmoHSP90 genes, MapChart software was used for mapping according to their chromosome information. The results indicated the distribution of CmoHSP90 genes on 9 out of 20 chromosomes of the pumpkin genome. Each chromosome contained only one CmoHSP90 gene, with the exception of the chr_16 chromosome that contained two genes (Figure 4A).
Gene duplication, which can be classified as tandem or segmental duplication, is an important feature of plant genomic structure and performs critical roles in the evolution of gene families. Therefore, we analyzed gene duplication of the CmoHSP90 gene in the pumpkin genome. A total of four pairs of CmoHSP90 genes, including CmoHSP90-1/CmoHSP90-10, CmoHSP90-2/CmoHSP90-7, CmoHSP90-3/CmoHSP90-6, and CmoHSP90-4/CmoHSP90-9, were determined to be segmental duplication (Figure 4B). The value of Ka/Ks (nonsynonymous/synonymous) can reflect the pressure of selection for a gene during evolution. Table 2 shows the Ka/Ks values of the duplicated genes calculated using TBtools, all of which were <1, suggesting that these CmoHSP90 genes were influenced by purifying selection during evolution. The estimated time of duplication for paralogous genes indicated that all paralogs were ancient (from 91 to 269 million years ago, Mya). The phylogenetic mechanisms of the CmoHSP90 family were explored by creating comparative syntenic maps of pumpkin related to other species (Figure 5 and Figure S3), and results showed that ten C. maxima HSP90 genes and ten pumpkin HSP90 genes were orthologous genes with 17 syntenic relationships, and two Arabidopsis HSP90 genes and three pumpkin HSP90 genes were orthologous genes with three syntenic relationships (Figure 5, Supplementary Table S5). Moreover, collinear gene pairs were identified in pumpkin and other Cucurbitaceous plants except monk fruit (Supplementary Figure S3). The collinear gene pair number between pumpkin and other Cucurbitaceous plants was greater than that between more distantly related Arabidopsis or rice, and no gene pairs were found between pumpkin and rice (Supplementary Table S5).

3.5. Analysis of CmoHSP90 Promoters

In all, 23 types of cis-acting elements in the promoter regions of CmoHSP90s were identified and classified into four categories: (1) hormone response elements including TCA-element, TGACG-motif, CGTCA-motif, TATC-box, P-box, AuxRR-core, TGA-element, GARE-motif, and ABRE; (2) stress response elements including WUN-motif, MBS, TC-rich repeats, and LTR; (3) developmental elements including MBSI, CAT-box, GCN4-motif, Circadian, and O2-site; and (4) light-responsive elements including I-box, Box 4, G-box, GT1-motif, and MRE (Figure 6A). The number of abscisic acid responsiveness (ABRE) and MeJA-responsiveness elements (TGACG-motif and CGTCA-motif) in hormone response elements was large. All CmoHSP90 gene promoters except for CmoHSP90-5 contained ABRE elements, among which the CmoHSP90-3 and CmoHSP90-6 promoters had the largest number (Figure 6B). The number of drought-inducibility elements (MBS) in the stress response elements was the largest, which existed in the promoters of CmoHSP90-3, CmoHSP90-8, CmoHSP90-9, and CmoHSP90-10. The number of developmental response elements was small, and the promoter sequences of CmoHSP90-1, CmoHSP90-5, CmoHSP90-8, and CmoHSP90-9 did not contain developmental response elements, while other promoter sequences contained only one to two developmental response elements. Among the light response elements, Box 4 and G-box had the largest number, and the promoter regions of all 10 CmoHSP90 genes contained Box 4 elements, while only CmoHSP90-5 and CmoHSP90-7 promoter regions did not contain the G-box elements (Supplementary Table S6).

3.6. Expression Profiling of CmoHSP90 Genes under Heat and Cold Stresses

The expression levels of 10 pumpkin CmoHSP90 genes in roots and leaves under low (4 °C) and high temperature (42 °C) were detected using RT-qPCR, and the results showed that under high-temperature treatment, all CmoHSP90 genes in leaves were up-regulated, and all genes except CmoHSP90-1 and CmoHSP90-8 in roots also exhibited up-regulated expression (Figure 7A). The expression level of CmoHSP90-3 in leaves was 151.43 times that of the control after a 6-h high-temperature treatment, and the level of CmoHSP90-6 was 122.37 times that of the control after a 3-h high-temperature treatment. The expression of CmoHSP90-6 in roots was 55.90 times that of the control after a 3-h high-temperature treatment. In contrast, the expression levels of CmoHSP90 genes did not significantly change after low-temperature treatment (Figure 7B). The expression of CmoHSP90-6 and CmoHSP90-10 in roots reached the highest levels after a 12-h and 3-h low-temperature treatment, which was 10.82 times and 14.24 times that of control, respectively. The expression of the CmoHSP90-8 gene in leaves reached the highest level after a 6-h low-temperature treatment, which was 4.47 times that of the control.

3.7. Protein-Protein Interaction Network

A full network map of selected CmoHSP90 and related genes in Arabidopsis was drawn using the STRING software to predict their physical and functional interactions. A total of 18 proteins were predicted in the network (Figure 8); these proteins were closely correlated with each other in biological processes. For instance, SHD protein, homologous of CmoHSP90-3 and CmoHSP90-6, has associations with HSP70, CRT3, SGT1A, SGT1B, and CRT1a. HSP90.1, the homologue of CmoHSP90-8, has associations with HSP81-3, ROF1, HSFA2, HSP70, SGT1A, SGT1B, SQN, and HOP3 proteins. The annotation and sequence information is listed in Supplementary Tables S7 and S8.

4. Discussion

The pumpkin is one of the most productive crops in the world and has the reputation of ’world vegetable‘. Pumpkin is mainly grown in tropical and subtropical areas, with strong heat resistance [26,27]. Therefore, studying the heat resistance mechanism of pumpkin can provide a good genetic resource for improving the heat resistance of vegetable crops. The HSP90 protein plays an important role in plant response to temperature stress [5,6]. With the release of various plant genome data, the HSP90 gene family has been identified in more and more vegetable crops such as pepper [38], cucumber [31], and tomato [39], as well as some model plants such as rice [11] and Arabidopsis [40]. However, to date, the HSP90 gene family has not been reported in pumpkin.
In the current study, we identified 10 HSP90 genes from the pumpkin genome, more than those identified in Arabidopsis (7 HSP90 genes) [40], pepper (7 HSP90 genes) [38], tomato (7 HSP90 genes) [39], and rice (9 HSP90 genes) [11]. The different number of HSP90 genes in different plant species might lead to different heat resistance in different plant species. The HSP90 gene family of 11 other Cucurbitaceous crops including cucumber, bitter gourd, wax gourd, melon, bottle gourd, Cucurbita maxima, watermelon, sponge gourd, chayote, snake gourd, and monk fruit were also downloaded according to a previous study [31] or identified in this study (Supplementary Table S2). The number of HSP90 members in other Cucurbitaceous crops (5 to 9 members) was less than that in pumpkin (C. moschata), except for Cucurbita maxima (10 HSP90 genes), which belongs to the Cucurbita genus as pumpkin. This may be the reason why C. moschata has high heat tolerance and is frequently utilized as rootstock for improving stress tolerance of cucurbits in production [29]. Physicochemical property analysis of CmoHSP90 proteins revealed a large diversity among these genes. All 10 pumpkin CmoHSP90 proteins except CmoHSP90-10 were acidic (Table 1), similar to those in other plants [31], suggesting that CmoHSP90-10 is different from other genes in evolution. The 100 HSP90 proteins from rice, Arabidopsis, and another 12 Cucurbitaceous crops analyzed using phylogenetic trees were classified into four groups: A, B, C, and D. HSP90 proteins from the 14 species except watermelon, bottle gourd, and monk fruit were all classified into the four groups. Group B did not contain the members from monk fruit, and group C did not contain the members from watermelon and bottle gourd (Figure 1 and Figure S1). These results indicate that most HSP90 proteins are conserved in different plant species. A conserved motif and similar exon/intron structure were observed for most of the HSP90 genes in each group, according to the gene structure and phylogenetic analysis results (Figure 2). Members from groups C and D contained fewer introns, while groups A and B contained more introns. Group A exhibited remarkable differences in gene structure and motif composition, suggesting that gene evolution event might affect both their functions and structures [41,42]. The different gene structure between different groups and similar gene structure in the same group were also observed in other Cucurbitaceous crop HSP90 family genes [31].
Tandem duplication, segmental duplication, and whole genome duplication (WGD) contribute to gene family expansion in plants [43,44]. In the pumpkin HSP90 gene family, four pairs of predicted segmentally duplicated genes were found, while tandemly duplicated genes were not discovered (Figure 4B), indicating that this gene family evolution has been strongly marked by species-specific gene duplication. The estimated time of duplication for the paralogous CmoHSP90 genes (91-269 Mya, Table 2) indicated that all paralogs occurred before diploid progenitor of pumpkin diverged from Benincaseae (about 26 Mya) and its allotetraploidization (about 3 Mya) [45]. These results suggest that the segmentally duplicated events are a key driving force for the expansion of pumpkin HSP90 family genes. Furthermore, the four pairs of segmentally duplicated genes were observed to undergo strong purifying selection pressure (Table 2), indicative of a highly conserved evolutionary pattern of pumpkin HSP90 genes. From the synteny analysis results of Chinese pumpkin HSP90 family genes with model plants rice and Arabidopsis, as well as 11 other Cucurbitaceous crops (Figure 5 and Figure S3), only two Arabidopsis HSP90 genes and three pumpkin HSP90 genes were orthologous genes with three syntenic relationships, while more syntenic relationships (7 to 17) were detected between pumpkin HSP90 genes and the Cucurbitaceous crops, and the most orthologous genes were found with the C. maxima HSP90 gene family, indicating the HSP90 gene family has a specific expansion mode in each species. It is surprising that no syntenic relationship was observed between pumpkin and monk fruit (data not shown). This is probably because one is a fruit tree and the other is a vegetable, and they have evolved differently. The monk fruit has a higher classification status in the Cucurbitaceae family, and the genetic relationship between the monk fruit and Cucurbitaceae supports the monk fruit as an independent genus using ITS2 and LEAFY intron 2 phylogenetic tree analyses [46].
Furthermore, as demonstrated by promoter analysis, the promoter regions of pumpkin HSP90 genes contained different regulatory elements including stress response, hormone response, light-responsive, and development-related elements, suggesting these genes are involved in protein trafficking and degradation, cell-cycle control, and signal transduction [47,48]. Plant hormones play crucial roles in response to heat stress and other environmental stresses, as well as plant development and growth [49]. ABA treatment or heat stress can induce LpHSP90-5 [50]. Heat stress can induce six HSP90 genes, which play critical roles in GA signaling [31]. Auxin can induce the sHSP22 heat shock protein, which can coordinate the auxin/ABA interaction and signaling in A. thaliana [51]. These results indicate that HSP expression is closely correlated with hormones. The hormone-related elements are commonly discovered in the promoter region of CmoHSP90 genes (Figure 6 and Supplementary Table S6). Therefore, even though there are no cold- or heat-responsive cis-elements in CmoHSP90 gene promoter regions, their expression can still be induced. After heat stress treatment, the expression of all the CmoHSP90 genes increased as detected by RT-qPCR, among which CmoHSP90-3 and CmoHSP90-6 increased hundreds of times after 3-6 h of heat stress treatment. Meanwhile, CmoHSP90-6 and CmoHSP90-10 were induced significantly after cold stress. These genes may be involved in the biological pathway of heat or cold stress.
Protein interaction network prediction is often used to study the function of genes [52]. As shown in Figure 8, Arabidopsis SGT1A, SGT1B, HSFA2, and HSP70 were predicted to be important for the functional partners of CmoHSP90s, because they occupied central positions in the protein-protein interaction network. Arabidopsis SGT1A and SGT1B, interacting with SHD (the homologue of CmoHSP90-3 and CmoHSP90-6), play a role in plant resistance against pathogens and are redundant in function [53]. SGT1 could associate with HSP90 and assisted HSP90 in stabilizing plant disease resistance proteins [53,54,55]. HSFA2 is strongly induced by heat and responsive to environmental stress including salt stress [56] and oxidative stress [57]. HSP70 could cooperate with other chaperones to assist refolding of misfolded stress-denatured proteins and prevent protein aggregation, thus controlling cell homeostasis, proliferation, differentiation, and cell death [58].

5. Conclusions

In this study, 10 CmoHSP90 genes were identified in the pumpkin genome and characterized using phylogenetic analyses, physicochemical properties, gene structure, conserved motifs, chromosomal location, homologous gene pairs, synteny, and cis-elements in the gene promoters, which revealed the evolutionary relationship of CmoHSP90 family genes. The expression of CmoHSP90 genes under heat and cold stresses was detected using RT-qPCR, and CmoHSP90-3 and CmoHSP90-6 were found to be significantly induced by heat stress, and CmoHSP90-6 and CmoHSP90-10 were significantly induced by cold stress. These results suggest that these genes play critical roles in acquisition of thermotolerance or cold acclimation in pumpkins. Our findings provide a foundation for understanding the CmoHSP90 gene family functions and may be helpful in breeding new pumpkin varieties with stress tolerance.

Supplementary Materials

The following supporting information can be downloaded at https://www.mdpi.com/article/10.3390/agronomy13020430/s1, Figure S1: distribution of members in each HSP90 group of different plant species, Figure S2: the amino acid sequences of each motif identified in CmoHSP90 proteins, Figure S3: synteny analysis of HSP90 genes in C. moschata and other Cucurbitaceous crops, Table S1: the primers used in this study, Table S2: the gene ID of HSP90 in different plant species, Table S3: the sequence of motifs in the CmoHSP90 protein, Table S4: the motifs and number of introns in each CmoHSP90 gene, Table S5: the collinear gene pairs between CmoHSP90 and other species, Table S6: information on cis-elements of CmoHSP90 gene promoters, Table S7: information on the network proteins, Table S8: sequences of the network proteins.

Author Contributions

Conceptualization, W.Z. and Y.Z.; formal analysis, Y.H., T.Z., P.W., Y.L., M.W., B.Z., D.L., T.Y., W.H., Y.C. and Y.Z.; funding acquisition, W.Z., T.Y. and Y.Z.; investigation, Y.H., T.Z., P.W., Y.L., M.W., B.Z., D.L., T.Y., W.H., Y.C. and Y.Z.; methodology, Y.H. and T.Z.; supervision, Y.Z.; writing—original draft preparation, Y.H., T.Z., W.Z. and Y.Z.; writing—review and editing, Y.Z. All authors have read and agreed to the published version of the manuscript.

Funding

This work was financially supported by the Major Science and Technology Plan of Hainan Province (ZDKJ2021005), Hainan Province Science and Technology Special Fund (ZDYF2021XDNY292), and the Opening Project Fund of Key Laboratory of Vegetable Biology of Hainan Province (HAAS2022PT0105).

Institutional Review Board Statement

Not applicable.

Informed Consent Statement

Not applicable.

Data Availability Statement

Not applicable.

Conflicts of Interest

The authors declare no conflict of interest.

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Figure 1. Phylogenetic analysis of HSP90 proteins from Cucurbita moschata, melon (Cucumis melo), cucumber (Cucumis sativus), Cucurbita maxima, bottle gourd (Lagenaria siceraria), wax gourd (Benincasa hispida), bitter gourd (Momordica charantia), sponge gourd (Luffa cylindrica), watermelon (Citrullus lanatus), chayote (Sechium edule), snake gourd (Trichosanthes anguina), monk fruit (Siraitia grosvenorii), Arabidopsis thaliana, and rice (Oryza sativa). A phylogenetic tree of HSP90 proteins was constructed using MEGA-X software. The four groups are indicated by different colors. The red stars represent Cucurbita moschata HSP90s, the black stars represent cucumber HSP90s, the blue stars represent melon HSP90s, the olive stars represent watermelon HSP90s, the teal stars represent C. maxima HSP90s, the peru stars represent bottle gourd HSP90s, the orange stars represent wax gourd HSP90s, the cyan stars represent sponge gourd HSP90s, the green stars represent bitter gourd HSP90s, the gold stars represent A. thaliana HSP90s, the purple stars represent rice HSP90s, the indigo stars represent chayote HSP90s, the pink stars represent snake gourd HSP90s, and the dodger blue stars represent monk fruit HSP90s.
Figure 1. Phylogenetic analysis of HSP90 proteins from Cucurbita moschata, melon (Cucumis melo), cucumber (Cucumis sativus), Cucurbita maxima, bottle gourd (Lagenaria siceraria), wax gourd (Benincasa hispida), bitter gourd (Momordica charantia), sponge gourd (Luffa cylindrica), watermelon (Citrullus lanatus), chayote (Sechium edule), snake gourd (Trichosanthes anguina), monk fruit (Siraitia grosvenorii), Arabidopsis thaliana, and rice (Oryza sativa). A phylogenetic tree of HSP90 proteins was constructed using MEGA-X software. The four groups are indicated by different colors. The red stars represent Cucurbita moschata HSP90s, the black stars represent cucumber HSP90s, the blue stars represent melon HSP90s, the olive stars represent watermelon HSP90s, the teal stars represent C. maxima HSP90s, the peru stars represent bottle gourd HSP90s, the orange stars represent wax gourd HSP90s, the cyan stars represent sponge gourd HSP90s, the green stars represent bitter gourd HSP90s, the gold stars represent A. thaliana HSP90s, the purple stars represent rice HSP90s, the indigo stars represent chayote HSP90s, the pink stars represent snake gourd HSP90s, and the dodger blue stars represent monk fruit HSP90s.
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Figure 2. Conserved motifs and gene structures of CmoHSP90 family members. (A) Conserved motifs of CmoHSP90 proteins. Different motifs are represented by colored boxes and different numbers. (B) Exon/intron structures of CmoHSP90 genes. Exons, introns, and UTRs are represented by yellow boxes, black lines, and green boxes, respectively. The phylogenetic tree, conserved motifs, and gene structures were visualized by TBtools.
Figure 2. Conserved motifs and gene structures of CmoHSP90 family members. (A) Conserved motifs of CmoHSP90 proteins. Different motifs are represented by colored boxes and different numbers. (B) Exon/intron structures of CmoHSP90 genes. Exons, introns, and UTRs are represented by yellow boxes, black lines, and green boxes, respectively. The phylogenetic tree, conserved motifs, and gene structures were visualized by TBtools.
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Figure 3. Amino acid sequence alignments of CmoHSP90 proteins. Red lines represent the ATP/ADP-binding sites. Red boxes mark conserved motifs and α-helix.
Figure 3. Amino acid sequence alignments of CmoHSP90 proteins. Red lines represent the ATP/ADP-binding sites. Red boxes mark conserved motifs and α-helix.
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Figure 4. Chromosome distribution and syntenic analysis of CmoHSP90 genes in pumpkin. (A) Chromosomal locations of CmoHSP90 genes. The chromosome numbers are listed at the top of each chromosome, and the number to the left of each chromosome represents the location of the CmoHSP90 gene on the right. Only the chromosomes where CmoHSP90 genes were mapped are shown. (B) Syntenic relationships of HSP90 gene family in pumpkin. Gray lines indicate synteny blocks within the pumpkin genome, and colored lines between CmoHSP90 genes represent segmental duplication events that occurred in the pumpkin HSP90 gene family.
Figure 4. Chromosome distribution and syntenic analysis of CmoHSP90 genes in pumpkin. (A) Chromosomal locations of CmoHSP90 genes. The chromosome numbers are listed at the top of each chromosome, and the number to the left of each chromosome represents the location of the CmoHSP90 gene on the right. Only the chromosomes where CmoHSP90 genes were mapped are shown. (B) Syntenic relationships of HSP90 gene family in pumpkin. Gray lines indicate synteny blocks within the pumpkin genome, and colored lines between CmoHSP90 genes represent segmental duplication events that occurred in the pumpkin HSP90 gene family.
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Figure 5. Synteny analysis of HSP90 genes in C. moschata, A. thaliana, and C. maxima. Gray lines indicate significantly collinear blocks within and among plant genomes, while red lines highlight syntenic HSP90 gene pairs. The numbers at the top or bottom of each chromosome represents the chromosome name.
Figure 5. Synteny analysis of HSP90 genes in C. moschata, A. thaliana, and C. maxima. Gray lines indicate significantly collinear blocks within and among plant genomes, while red lines highlight syntenic HSP90 gene pairs. The numbers at the top or bottom of each chromosome represents the chromosome name.
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Figure 6. Cis-element analysis of the CmoHSP90 gene promoter regions. (A) Colored blocks represent different types of cis-elements and their locations in each CmoHSP90 gene promoter. (B) The different colors and numbers indicate the numbers of different promoter elements in the CmoHSP90 gene promoters. The types, numbers, and locations of potential elements in the promoter regions 2.0-kb upstream of the CmoHSP90 genes were determined using PlantCARE software.
Figure 6. Cis-element analysis of the CmoHSP90 gene promoter regions. (A) Colored blocks represent different types of cis-elements and their locations in each CmoHSP90 gene promoter. (B) The different colors and numbers indicate the numbers of different promoter elements in the CmoHSP90 gene promoters. The types, numbers, and locations of potential elements in the promoter regions 2.0-kb upstream of the CmoHSP90 genes were determined using PlantCARE software.
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Figure 7. Expression analysis of the pumpkin CmoHSP90 genes in response to heat (A) and cold (B) stresses using RT-qPCR. The mean expression value was calculated from three replicates. Vertical bars represent means of fold change in expression and standard deviations calculated from replicates. Values of 0, 3, 6, 9, 12, and 24 indicate hours after treatment. Asterisk (* or **) indicate a significant difference at p < 0.05 or 0.01, respectively.
Figure 7. Expression analysis of the pumpkin CmoHSP90 genes in response to heat (A) and cold (B) stresses using RT-qPCR. The mean expression value was calculated from three replicates. Vertical bars represent means of fold change in expression and standard deviations calculated from replicates. Values of 0, 3, 6, 9, 12, and 24 indicate hours after treatment. Asterisk (* or **) indicate a significant difference at p < 0.05 or 0.01, respectively.
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Figure 8. Protein-protein interaction (PPI) networks of CmoHSP90s and their related proteins. Each node represents all the proteins produced by a single protein-coding gene locus. The colored nodes represent the query proteins and first shell of interactors. Edges indicate both functional and physical protein associations. The colored lines between the nodes indicate the type of interaction. The known interactions use light blue lines (from curated databases) and purple lines (experimentally determined). The predicated interactions use green (gene neighborhood), red (gene fusions), and blue lines (gene co-occurrence). The yellow, black, and cyan lines represent the text mining, coexpression, and protein homology, respectively.
Figure 8. Protein-protein interaction (PPI) networks of CmoHSP90s and their related proteins. Each node represents all the proteins produced by a single protein-coding gene locus. The colored nodes represent the query proteins and first shell of interactors. Edges indicate both functional and physical protein associations. The colored lines between the nodes indicate the type of interaction. The known interactions use light blue lines (from curated databases) and purple lines (experimentally determined). The predicated interactions use green (gene neighborhood), red (gene fusions), and blue lines (gene co-occurrence). The yellow, black, and cyan lines represent the text mining, coexpression, and protein homology, respectively.
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Table
Table 1. Physicochemical properties of CmoHSP90 proteins in the pumpkin genome.
Table 1. Physicochemical properties of CmoHSP90 proteins in the pumpkin genome.
Gene NameGene IDLocusLength (aa)MW (kDa)pINo. of TransmembraneSubcellular Localization
CmoHSP90-1CmoCh02G002280Chr02: 1063366..107296756764.32 6.24-mitochondrial
CmoHSP90-2CmoCh04G022830Chr04: 17001131..1700824579389.91 4.89-mitochondrial
CmoHSP90-3CmoCh05G003160Chr05: 1403562..140821881693.26 4.92-endoplasmic reticulum
CmoHSP90-4CmoCh06G007160Chr06: 3650095..365314870080.02 4.97-cytoplasmic, nuclear
CmoHSP90-5CmoCh11G012410Chr11: 7760957..776373370180.22 4.99-cytoplasmic
CmoHSP90-6CmoCh12G003530Chr12: 2180257..218512581393.16 4.95-endoplasmic reticulum
CmoHSP90-7CmoCh15G008820Chr15: 4464899..447094683294.64 4.95-mitochondrial
CmoHSP90-8CmoCh16G001050Chr16: 480853..48367270480.87 4.95-cytoplasmic, nuclear
CmoHSP90-9CmoCh16G010310Chr16: 7195032..719832970080.26 4.97-cytoplasmic
CmoHSP90-10CmoCh20G006750Chr20: 3297642..330958286597.36 8.222endoplasmic reticulum
Table
Table 2. Ka/Ks values of CmoHSP90 duplicated genes.
Table 2. Ka/Ks values of CmoHSP90 duplicated genes.
Gene Name_1Gene Name_2KaKsKa/KsData (Mya) a
CmoHSP90-1CmoHSP90-100.4498322380.9678774720.464761554269
CmoHSP90-2CmoHSP90-70.0343033150.3281003910.10455127791
CmoHSP90-3CmoHSP90-60.033771630.3953404340.085424175110
CmoHSP90-4CmoHSP90-90.0145967810.5488925040.026593151152
a The divergence time was estimated according to formula T = Ks/2λ. The clock-like rate (λ) was 1.8 × 10−9 substitutions per site per year. Mya, million years ago.
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Hu, Y.; Zhang, T.; Wang, P.; Li, Y.; Wang, M.; Zhu, B.; Liao, D.; Yun, T.; Huang, W.; Chen, Y.; et al. Genome-Wide Characterization of HSP90 Gene Family in Chinese Pumpkin (Cucurbita moschata Duch.) and Their Expression Patterns in Response to Heat and Cold Stresses. Agronomy 2023, 13, 430. https://doi.org/10.3390/agronomy13020430

AMA Style

Hu Y, Zhang T, Wang P, Li Y, Wang M, Zhu B, Liao D, Yun T, Huang W, Chen Y, et al. Genome-Wide Characterization of HSP90 Gene Family in Chinese Pumpkin (Cucurbita moschata Duch.) and Their Expression Patterns in Response to Heat and Cold Stresses. Agronomy. 2023; 13(2):430. https://doi.org/10.3390/agronomy13020430

Chicago/Turabian Style

Hu, Yanping, Tingting Zhang, Peng Wang, Yuxin Li, Min Wang, Baibi Zhu, Daolong Liao, Tianhai Yun, Wenfeng Huang, Yisong Chen, and et al. 2023. "Genome-Wide Characterization of HSP90 Gene Family in Chinese Pumpkin (Cucurbita moschata Duch.) and Their Expression Patterns in Response to Heat and Cold Stresses" Agronomy 13, no. 2: 430. https://doi.org/10.3390/agronomy13020430

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