Open AccessArticle

Comprehensive Genomic Analysis and Expression Profile of Hsp70 Gene Family Related to Abiotic and Biotic Stress in Cucumber

Zixian Zhou

^1,2,†,

Lingdi Xiao

^1,†,

Jindong Zhao

¹,

Zhaoyang Hu

¹,

Yuelong Zhou

Shiqiang Liu

Hao Wu

^2,3,* and

Yong Zhou

^1,4,*

College of Bioscience and Bioengineering, Jiangxi Agricultural University, Nanchang 330045, China

Guangdong Provincial Key Laboratory of Utilization and Conservation of Food and Medicinal Resources in Northern Region, Shaoguan University, Shaoguan 512000, China

School of Biology and Agriculture, Shaoguan University, Shaoguan 512005, China

⁴

Key Laboratory of Crop Physiology, Ecology and Genetic Breeding, Ministry of Education, Jiangxi Agricultural University, Nanchang 330045, China

Authors to whom correspondence should be addressed.

^†

These authors contributed equally to this work.

Horticulturae 2023, 9(9), 1057; https://doi.org/10.3390/horticulturae9091057

Submission received: 26 August 2023 / Revised: 16 September 2023 / Accepted: 18 September 2023 / Published: 21 September 2023

(This article belongs to the Special Issue Recent Scientific Developments in Genetic Improvement of Vegetables for Resistance to Biotic and Abiotic Stresses)

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Figure 1
Phylogenetic tree of Hsp70 proteins in cucumber (Cucumis sativus), Arabidopsis (Arabidopsis thaliana), rice (Oryza sativa), pepper (Capsicum annuum), maize (Zea mays), and tomato (Solanum lycopersicum). Four groups (A–D) are represented by different colors, and CsHsp70s are represented by stars. The accession numbers of Hsp70 proteins are provided in <a href="#app1-horticulturae-09-01057" class="html-app">Supplementary Table S2</a>. "> Figure 2
Conserved motif analysis of CsHsp70 proteins on the basis of the phylogenetic relationship. Ten motifs were marked by different colors and length of box denotes motif length. "> Figure 3
Distribution of the CsHsp70 genes on the five chromosomes of cucumber. The bar located on the left side representing the chromosome length was showed in megabase (Mb), and segmental duplication genes are marked with red and connected with red lines. "> Figure 4
Phytohormone- and stress-responsive cis-elements in CsHsp70s promoter regions. The amounts of cis-elements in CsHsp70s promoter regions were displayed as different colors and numbers in the grid. "> Figure 5
The transcription levels of CsHsp70 genes under salt stress (A), different temperature and photoperiod combined conditions (B) based on RNA-seq data. The red to blue colors on the scale located on the right side representing high to low gene expression, which were calculated as the log2(TPM+1) values. CK-L and Na-L, leaf sample from control and salt-stressed plants; CK-R and Na-R, root sample from control and salt-stressed plants. HL and LL, high and low temperature under long day; HS and LS, high and low temperature under short day. "> Figure 6
Transcription levels of five selected CsHsp70 genes under drought (A) and ER stress (B) treatments, as examined using qRT-PCR. Asterisks indicate significant differences examined by the student’s t-test (p < 0.05). "> Figure 7
Heat map of differential transcription of CsHsp70 genes under inoculation of PM (Sphaerotheca fuliginea) (A), RKN (Meloidogyne incognita) (B), and DM (Pseudoperonospora cubensis) (C). The transcriptional levels of CsHsp70 genes are displayed as log2 transformed TPM values. Different colors on the scale located on the right side represent differential gene expression, which decrease from red to blue. ">

Versions Notes

Abstract

Heat shock protein 70 (Hsp70) is a class of HSPs involved in plant growth and development, stress response and regulation. The Hsp70 proteins exist widely in the plant world, but the detail information about Hsp70s is still unclear in cucumber. Based on the available cucumber genome, a total of 12 Hsp70 genes (CsHsp70-1 to CsHsp70-12) were identified in this study, and they were distributed among five out of seven chromosomes. The CsHsp70s were divided into four groups based on a phylogenetic analysis by using protein sequences from cucumber and other plants, and their conserved motifs were relatively conserved. Gene duplication analysis showed that segmental duplication is the main driving force of expansion in cucumber CsHsp70 genes. Promoter analysis of CsHsp70 genes showed that they contained many cis-acting elements involved in hormone and stress responses. Expression analysis by RNA-seq and qRT-PCR indicated that the expression of most CsHsp70 genes was associated with multiple biotic and abiotic stresses in cucumber. This study introduces the characteristics of cucumber CsHsp70 genes and the regulation of their expression levels in various abiotic and biotic stresses, which provided a basis for functional exploration and utilization of CsHsp70 genes in the future.

Keywords:

cucumber; heat shock protein 70 (Hsp70); stress response; expression pattern; gene family

1. Introduction

Various abiotic and biotic stresses have huge impacts on the growth and development of crop plants, and thus reduce the yield and economic benefit. To deal with these adverse factors, plants produce quite a lot of induced proteins due to their ability to protect other proteins from denaturation or aggregation under stresses. Among these proteins, heat shock proteins (HSPs) are a class of molecular chaperones originally discovered according to their increased levels when cells are suffered by heat stress [1,2]. HSPs are highly conserved and widely distributed in both the prokaryotic and eukaryotic organisms, and they can be divided into five major sub-families, namely, HSP100s, HSP90s, HSP70s, HSP60s, and small HSPs (sHSPs), on the basis of their molecular weights (MWs) [3]. Among these families, HSP70s were considered to be the most evolutionarily conserved HSPs in every kingdom, from prokaryotes (DnaK) and eukaryotes (HSP70). The MW of HSP70s is mostly between 68–78 kDa, and all members of HSP70s comprise a highly conserved N-terminal nucleotide binding domain (NBD) for ATP-binding and a substrate binding domain (SBD) for binding to the substrate located in the C-terminal region [4]. The two domains are connected by an intermediate domain (about 15 kDa). In plants, four types of Hsp70s were widely distributed in different subcellular compartments: mitochondria, cytoplasm, chloroplast, and endoplasmic reticulum (ER) [5]. And Hsp70s in other subcellular compartments were also observed. For example, a tobacco nuclear-localized HSP70, NtHSP70-1, is involved in nuclear DNA stability for preventing the fragmentation and degradation during heat stress [6].

Plant Hsp70 genes were composed of a multi-gene family and have been extensively identified in a variety of plants, such as rice [7,8], pepper [9], potato [10], tobacco [11], cabbage [12], tomato [13,14], pumpkin [15], chrysanthemum [16], and radish [17]. In previous studies, many recognized Hsp70 genes have been characterized to quickly express and accumulate under a number of abiotic stresses, such as heavy metal [14,18], salt [19], drought [20,21], heat [7,16], UV and high intensity light stress [22], as well as biotic stresses, such as pathogen and virus infection [23,24]. These reports revealed the possible roles of Hsp70 genes in improving the tolerance of plants to various stresses. For example, overexpression of GhHSP70-26 markedly enhances drought resistance and reduces cell membrane damage and reactive oxygen species (ROS) accumulation in transgenic tobacco plants, whereas GhHSP70-26 silenced cotton plants displayed increased sensitivity to drought stress, indicating that GhHSP70-26 positively regulated drought stress in cotton [25]. Overexpression and silencing of RsHSP70-20 in radish cotyledons showed that RsHSP70-20 paly a positive role in regulating of heat stress response in radish [17]. Many adverse abiotic and biotic stress treatments (such as heat, salt stress and pathogen infection) can induce ER stress by affecting the accumulation of unfolded and misfolded proteins in the ER [26]. BiP (binding protein) is a subfamily of HSP70 and one of the most abundant chaperones involve in ER stress. There are three BiP genes in pepper, where overexpression of CaBiP1/CaHsp70-8 promoted tolerance to ER stress and a variety of environment stresses in transgenic Arabidopsis plants [27]. Additionally, the developmental role of the Hsp70 genes were also experimentally characterized in some plant species. For instance, Arabidopsis plants have a deficiency in AtHSP70-15 displayed severe growth retardation and accelerated wilting due to impaired stomatal closure [23]. OsHsp70cp-2, encoding a plastid-localized Hsp70, was involved in two Tic translocon components (Tic110 and Tic40) and important for amyloplast development in rice [28].

Cucumber (Cucumis sativus L.) is one of the most crucial vegetable crops, but its growth and development are frequently challenged by a number of unfavorable environmental conditions [29]. Since the Hsp70 gene family in cucumber have been identified in a previous report [30], the detail information, especially abiotic and biotic stress responses of CsHsp70 genes was still limited. In this study, we focus attention on the identification and characterization of Hsp70 family genes in cucumber, and systematically analyzed their evolutionary relationship, conserved motif, and cis-acting regulatory elements in their promoter regions. In addition, the expression patterns of CsHsp70 genes under multiple abiotic and biotic stresses were also determined by qRT-PCR and public RNA-seq data. The findings of our study provide valuable information for revealing the roles of CsHsp70 genes, which maybe serve as molecular resources for developing new resistant varieties for cucumber breeding.

2. Results

2.1. Genome-Wide Identification of the Hsp70 Family Genes in Cucumber

In total, 12 Hsp70 genes were identified in the cucumber genome, which were designated as CsHsp70-1 to CsHsp70-12 according to their location on the seven cucumber chromosomes (Table 1). The CDS lengths of CsHsp70 genes varied from 1716 bp (CsHsp70-3) to 2697 bp (CsHsp70-7), encoded proteins varied from 571 to 898 amino acids (aa) in length with MW values ranging from 61.99 to 100.14 kDa. Other physicochemical parameters of all CsHsp70 proteins, including pI and GRAVY values, were also examined. The pIs of the CsHsp70 proteins varied from 5.09 (CsHsp70-11) to 5.69 (CsHsp70-5), suggesting that all of them are acidic proteins. Besides, the GRAVYs of the CsHsp70s were varied from −0.498 (CsHsp70-7) to 0.040 (CsHsp70-3) (Table 1).

2.2. Evolutionary Relationships of CsHsp70 Proteins

To explore the evolutionary relationships of the CsHsp70 proteins, a phylogenetic tree was generated using the protein sequences of the Hsp70 family members from 12 CsHsp70s in cucumber, 18 AtHsp70s in Arabidopsis [31], 32 OsHsp70s in rice [8], 21 CaHsp70s in pepper [9], 22 ZmHsp70s in maize [32], and 21 SlHsp70s in tomato [13]. The resulting phylogenetic tree revealed that the above Hsp70 proteins were clustered into four groups (A–D) based on the bootstrap support (Figure 1). Group A was the largest group and contained the most Hsp70 proteins, while group B had the fewest. In addition, groups A and D covered the most numbers of CsHsp70s (four members each), while one and three CsHsp70s fell into groups B and C, respectively (Figure 1).

2.3. Conserved Motif Analysis of CsHsp70 Proteins

To study the evolutionary divergence of Hsp70 proteins in cucumber, the number and arrangement of conserved motifs were predicted with the MEME tool. As a result, we identified ten motifs and their details are shown in Supplementary Table S3. In addition, these motifs were widely found in the CsHsp70 proteins with several exceptions (Figure 2). For example, CsHsp70-2 in group C had an additional motif 7 in the N-terminus, while CsHsp70-10 in group A harbored an additional motif 9 in its C-terminus. Motifs 9, 4 and 6 were absent in CsHsp70-3, CsHsp70-7 and CsHsp70-11, respectively. Additionally, CsHsp70-4, CsHsp70-7 and CsHsp70-12 in group D missed motif 2, and both of CsHsp70-4 and CsHsp70-7 lacked motif 3 (Figure 2). These findings reflect the diversity and conservation among CsHsp70s.

2.4. Chromosomal Location and Gene Duplication Analysis of the CsHsp70 Genes

Chromosomal location analysis indicated that 12 CsHsp70 genes were distributed on five out of the seven chromosomes (Chrs) in cucumber genome (Figure 3). Chrs 4 and 5 had the highest number of CsHsp70 genes (three genes each), while Chrs 2, 3 and 7 each contained two CsHsp70 genes. Gene duplication event analysis displayed that two CsHsp70 genes, CsHsp70-6 and CsHsp70-10, were made up of one segmental duplication event (Figure 3).

2.5. Cis-Acting Regulatory Element Analysis of the CsHsp70 Genes

To investigate the potential functions of CsHsp70 genes, cis-acting regulatory elements of the promoter regions of CsHsp70 genes were examined. We divided the identified cis-elements into two categories: stress-responsive and phytohormone-responsive (Figure 4). Nine kinds of stress-responsive cis-elements were filtered out, including ARE involved in anaerobic induction, GC-motif involved in anoxia response, LTR involved in low-temperature response, MBS involved in drought-related response, STRE involved in stress-responsive, TC-rich repeats involved in defense and stress responses, W-box involved in fungal elicitor response, WRE3 and WUN-motif involved in wounding responses (Figure 4). Each CsHsp70 gene contained at least two kinds of stress-related cis-elements in their promoter regions, among which CsHsp70-4 had the largest number of stress-responsive elements. It should be noted that ARE involved in anaerobic induction were the most abundant cis-element and was present in all CsHsp70 promoter regions, particularly in CsHsp70-10, suggesting that the CsHsp70 genes are potentially regulated by anaerobic induction. In addition, many hormone-responsive cis-elements were identified in the CsHsp70 genes in response to distinct phytohormones, such as abscisic acid (ABA, including ABRE), auxin (including TGA-element and AuxRR-core), methyl jasmonate (MeJA, including CGTCA-motif and TGACG-motif), ethylene (including ERE), salicylic acid (SA, including TCA-element), and gibberellin (GA, including P-box, TATC-box and GARE-motif) (Figure 4). It is worth noting that the distribution pattern of hormone-responsive cis-elements differed among CsHsp70 genes. For example, ABRE was most abounding in the promoter regions of CsHsp70-7 and CsHsp70-8 (nine each), suggesting that they may mostly be associated with the ABA response, while CsHsp70-1, CsHsp70-5, CsHsp70-9 and CsHsp70-12 promoters contained none. P-box, TATC-box and GARE-motif were GA-responsive cis-elements, and they were unequally located in the 2, 4 and 2 CsHsp70 genes, respectively (Figure 4).

2.6. Expression Analysis of CsHsp70 Genes under Different Abiotic Stresses

To identify the roles of CsHsp70 genes in response to abiotic stress, the TPM values of CsHsp70 genes under salt, temperature, and photoperiod treatments were obtained by public RNA-seq data [33,34,35], and these transcription levels were assembled hierarchically in a heatmap. Under salt stress, the transcription levels of CsHsp70-6 and CsHsp70-9 were dramatically elevated in the leaf and root (Figure 5A). In addition, the transcription levels of CsHsp70-1, CsHsp70-3, CsHsp70-10, and CsHsp70-11 were observably increased in the leaf, while CsHsp70-5 and CsHsp70-8 exhibited significantly increased expression levels in the root (Figure 5A). Under different temperature and photoperiod combined conditions, the expression levels of CsHsp70-1, CsHsp70-10, and CsHsp70-11 were induced by high temperature under short days, while their transcription levels were repressed by high temperature under long days (Figure 5B), suggesting that they were involved in differential temperature-photoperiod environments.

To understand if CsHsp70 genes were regulated by drought and ER stress, the expression patterns of the seleted CsHsp70 genes under PEG and DTT conditions were measured by qRT-PCR. Under drought stress, CsHsp70-1 displayed a markedly up-regultaion expression from 2 h to 8 h, and its expression peaked at 8 h, while the other three CsHsp70 genes (CsHsp70-6, CsHsp70-8, CsHsp70-9 and CsHsp70-10) exhibited obviously decreased transcription levels at certain points under drought stress (Figure 6A). Under ER stress, the transcripts of CsHsp70-6, CsHsp70-8 and CsHsp70-9 were obviously increased at all test time points, while CsHsp70-1 display distinctly down-regultaion expression. CsHsp70-10 displayed an increased expression at the earlier time point (4 h), but its expression showed an obvious decline at the later time points (8 h and 12 h) (Figure 6B).

2.7. Expression Analysis of CsHsp70 Genes in Response to Different Biotic Stresses

A previous study showed that the expression levels of CsHsp70 genes were altered by the Pseudoperonospora cubensis infection [30], implying their roles in regulating biotic stress. To further understand whether CsHsp70 genes were responses to various biotic stresses, the expression profiles of CsHsp70 genes under three different biotic stresses, including PM (Sphaerotheca fuliginea), RKN (Meloidogyne incognita), and DM (Pseudoperonospora cubensis) were examined using the available RNA-seq data [36,37,38]. Upon the PM infection, the transcriptional levels of CsHsp70-6, CsHsp70-9 and CsHsp70-10 were increased in both resistant and susceptible cucumber cultivars (Figure 7A). CsHsp70-2, CsHsp70-5, CsHsp70-11, and CsHsp70-12 displayed increased transcriptional levels after PM treatment in the resistant cucumber line SSL508-28, while their expression was unaltered in the susceptible cucumber line D8 (Figure 7A). After the treatment with RKN inoculation, four CsHsp70 genes, CsHsp70-1, CsHsp70-3, CsHsp70-10 and CsHsp70-11, were found to be up-regulated in both resistant and susceptible cucumber cultivars (Figure 7B). We also determined the transcriptional levels of CsHsp70 genes from Vlaspik (DM susceptible line) and PI197088 (DM resistant line) after DM infection (Figure 7C). The results showed that CsHsp70-6, CsHsp70-7, CsHsp70-8, and CsHsp70-9 showed a markedly up-regulation trend after DM inoculation in both resistant and susceptible cucumber lines, suggesting their possible roles in DM invasion. Notably, CsHsp70-10 was down-regulated after the DM inoculation in Vlaspik (DM susceptible), while its expression was increased to a different extent in PI197088 (DM resistant) cucumber plants (Figure 7C). Therefore, some CsHsp70 genes might be associated with biotic stress responses in cucumber.

3. Discussion

Hsp70 proteins are widespread in every kingdom and mainly reported in heat shock responses. In this work, 12 Hsp70 genes were identified and systematically analyzed in cucumber. The protein sequences of the Hsp70s were highly conserved, but the number of Hsp70 family genes varied greatly in different plant species. For instance, the numbers of the Hsp70 gene family were 18 in Arabidopsis, 20 in potato [10], 21 in pepper [9], 21 in pumpkin [15], 24 in common bean [19], 32 in rice [7,8], 34 in radish [17], 52 in cabbage [12], 61 in tobacco [11], and 83 in chrysanthemum [16]. The reason for the different gene numbers of the above plant species may be due to the size of the genome. However, there was a disproportionate number of Hsp70 genes and a disproportionate genome size in several of the plant species, suggesting that the Hsp70 gene family has also undergone different duplication events. For example, three tandem and two segmented duplicated gene couples were observed among the 10 StHsp70 genes in potato [10]. Among the 52 BoHSP70 genes in cabbage [12], one tandem and 25 segmental duplication events were identified. Collinearity analysis of pumpkin Hsp70 genes showed that nine gene pairs were segmentally duplicated [15]. However, only two CsHsp70 genes made up to one segmental duplication event in the cucumber genome (Figure 3). Therefore, species–specific duplication or deletion during evolution may have resulted in differences in the numbers of the Hsp70 family genes across distinct plant species. In addition, the number of Hsp70s in different plants varied in each group (Figure 1), indicating that the roles of these Hsp70s belonging to different groups may undergo variations by environmental selection, resulting in the gain and/or loss of some functional genes.

According to the phylogenetic analysis, the Hsp70 family members can be clustered into four groups (Figure 1), which is consistent with the previous reports [4,8]. High sequence similarity of members in a gene family often indicates similar roles of them in different organisms. The CsHsp70s show closer phylogenetic relationships with the Hsp70s in dicots (Arabidopsis, pepper and tomato) than those in monocots (rice and maize), suggesting that Hsp70 genes might have similar functions in cucumber as their homologous genes in other plants. It is worth noting that each group comprised Hsp70s from the detected plants (Figure 1), implied that the Hsp70 gene family has undergone species-specific expansion during evolution in these plants. In addition, most CsHsp70s exhibit much similar conserved motif organization patterns (Figure 2), further supporting the relatively slow evolutionary process of the CsHsp70 genes. Nevertheless, further research to reveal the specific evolution process is still needed.

The determination of gene expression patterns provide insights into studying molecular functions of genes involved in diverse processes. It had been reported that most CsHsp70s were highly expressed in various cucumber tissues and also have differential expression under heat, salt, and P. cubensis infection, implying that they may take part in developmental processes of cucumber [30]. In the present study, many hormone- and stress-responsive cis-elements identified in the promoters of CsHsp70s revealed the involvement of them in cucumber stress tolerance (Figure 4). We thus further analyzed the transcriptional levels of CsHsp70 genes in response to distinct abiotic and biotic stresses. Our heatmap data showed that 8 out of 12 CsHsp70 genes exhibited significantly increased expression levels in leaf or root under salt stress (Figure 5A), implying that they have positive functions in the responses to salt stress. A previous report showed that overexpression of Erianthus arundinaceus EaHSP70 promoted tolerance of sugarcane plants to salt stress [39]. Expression of EsHSP70 by both constitutive and stress inducible promoters conferred tolerance to salt stress in transgenic Arabidopsis plants [40]. DTT is a redox reagent and act as an ER stress inducer by affecting disulfide bond formation, and previous study revealed that the expression levels of rice OsBiP4 and OsBiP5 were highly and specifically activated under DTT-induced ER stress [41]. In this study, the expression of selected five CsHsp70 genes responded to varying degrees under ER stress induced by DTT treatment, among which CsHsp70-8 extremely up-regulated (Figure 6B). CsHsp70-8 had HDEL (ER retention signal) in its C-terminus and it was clustered closely with CaBiP1/CaHsp70-8 and other BiPs in group D (Figure 1). Therefore, it can be speculated that CsHsp70-8 performed vital roles in ER stress and other environmental stresses. In addition, several CsHsp70 genes displayed altered expression under drought and differential temperature-photoperiod environments. Similar results were also observed in previous reports, such as potato [10], tomato [13], pumpkin [15], and chrysanthemum [16], in which a large proportion of Hsp70 genes display inducibility in response to at least one or more abiotic stresses.

Previous studies revealed that the expression levels of HSP70s can also respond to pathogen invasion, suggesting that they are an integral part of plant immunity [42,43]. In this study, PM, RKN and DM invasion observably altered the transcription levels of 7, 4 and 8 CsHsp70 genes, respectively (Figure 7). Notably, CsHsp70-6 and CsHsp70-9 displayed up-regulated expression levels in both susceptible and resistant cucumber lines under PM and DM treatments (Figure 7A,C), indicating that the two CsHsp70 genes might act as positive regulators in resistance to S. fuliginea and P. cubensis infection. In addition, the expression of CsHsp70-10 displayed increased levels in both susceptible and resistant cucumber lines under PM and RKN treatments, while its expression levels were opposite between resistant and susceptible cucumber plants under DM treatment (Figure 7). The changes in Hsp70 expression observed in this study suggested that some CsHsp70 genes play important roles in response to pathogen invasion of cucumber.

4. Materials and Methods

4.1. Database Searches and Annotation of the Hsp70 Members in Cucumber

To identify Hsp70 family members in cucumber, we used the Hidden Maekov Model (HMM) profile of the Hsp70 domain (PF00012) acquired from the Pfam database (http://pfam.xfam.org/, accessed on 1 May 2023) for hmmsearch (HMMER version 3.3.2) against the Cucurbit Genomics Database (CuGenDB, http://cucurbitgenomics.org/, accessed on 1 May 2023). In addition, the full-length Hsp70 amino acid sequences from rice [8], pepper [9], and tomato [13], were also retrieved and used as query sequences to perform BLAST (basic local alignment search tool) search against the CuGenDB database. The resulting candidate cucumber Hsp70s were confirmed by SMART (http://smart.embl-heidelberg.de/, accessed on 1 May 2023), for the presence of the Hsp70 domain (PF00012). The physicochemical properties of CsHsp70 proteins, including molecular weight (MW), isoelectric point (pI), and GRAVY (grand average of hydropathicity), were predicted using ProtParam server on ExPASy (http://web.expasy.org/protparam/, accessed on 1 May 2023).

4.2. Phylogenetic Analysis and Conserved Motif Identification

Multiple sequence alignments were conducted with the MAFFT tool (https://www.ebi.ac.uk/Tools/msa/mafft/, accessed on 1 May 2023) by using full-length protein sequences of Hsp70s from different plant species, and a Neighbor-Joining (NJ) phylogenetic tree was generated with MEGA 7.0 software using a bootstrap option of 1000 replications. MEME (http://meme-suite.org/tools/meme/, accessed on 1 May 2023) was used to predict the conserved motifs of CsHsp70s with the default settings, with the exception of the maximum number of diverse motifs was set as 10.

4.3. Prediction of Chromosomal Locations, Gene Duplication, and Cis-Acting Regulatory Elements of CsHsp70 Genes

The chromosomal locations of CsHsp70 genes were obtained from CuGenDB and drawn with the MG2C tool (http://mg2c.iask.in/mg2c_v2.1/, accessed on 1 May 2023) according to a previous report [29]. Gene duplication analysis was performed with the MCScanX software. The 2000 bp sequences in the 5′ flanking region from the promoters of CsHsp70 genes were extracted from CuGenDB, and then the PlantCARE tool (https://bioinformatics.psb.ugent.be/webtools/plantcare/html/, accessed on 1 May 2023) was used to determine the cis-acting regulatory elements present in the putative promoters of CsHsp70 genes.

4.4. Expression Analysis of the CsHsp70 Genes via RNA-Seq Data

To analyze the expression patterns of CsHsp70 genes under various stresses, the RNA-seq data were obtained from NCBI database with the BioProject IDs of PRJNA285071 (downy mildew, DM) [36], PRJNA321023 (powdery mildew, PM) [37], PRJNA419665 (root-knot nematode, RKN) [38], and PRJNA380322 (temperature and photoperiod) [33]. The RNA-seq data of cucumber under salt stress (CK-L and Na-L were represented as the leaf sample from control and salt-stressed plants, while CK-R and Na-R were represented as the root sample from control and salt-stressed plants) were also downloaded under the BioProject IDs of PRJNA477930 and PRJNA511946 [34,35]. The expression levels of CsHsp70 genes were calculated as transcripts per million reads (TPM) on the basis of the methods in previous studies [29,44]. The expression heatmaps were visualized with TBtools [45].

4.5. Plant Materials and Stress Treatments

Chinese Long cucumber inbred line 9930 seedlings were treated with drought and ER stress. In brief, two-leaf stage cucumber seedlings were transferred to Hoagland nutrition solution containing PEG-6000 (10%) and 2 mm dithiothreitol (DTT, a reducing agent that blocks the formation of disulfide bridge), for drought stress and endoplasmic reticulum (ER) stress, respectively. The leaf samples were harvested for each stress at various time points (0, 4, 8 and 12 h) and frozen with liquid nitrogen. Each stress was completed with three biological replicates.

4.6. RNA Extraction and Quantitative Real-Time PCR (qRT-PCR) Analysis of the CsHsp70 Genes

Total RNA was isolated using the RNAsimple Total RNA Kit (Tiangen, Beijing, China), and cDNA was synthesized for qRT-PCR by the HiScript^® III RT SuperMix for qPCR (Vazyme, Nanjing, China). The qRT-PCR was carried out on the Roche LightCycler 480 (LC480) system using TransStart Top Green qPCR SuperMix (+Dye II) (TransGen, Beijing, China) with three independent replicates. The amplification reaction included an initial 30 s denaturation at 95 °C, subsequently 40 cycles of 95 °C for 10 s and 60 °C for 30 s. The relative transcriptional levels of the CsHsp70 genes were calculated by using the cucumber β-Actin gene (Csa2G301530) as an internal control. The 2^−ΔΔCt method was implemented to calculate the relative gene transcriptional level of CsHsp70 genes, among which the samples at 0 h were set as 1.0. The qRT-PCR primers were shown in Supplementary Table S1.

5. Conclusions

In the present work, we identified 12 CsHsp70 genes from the C. sativus genome. The proteins encoded by these genes contained different amounts of amino acids and ranged from 61.99 to 100.14 kDa in size. The analyses of evolutionary relationship, conserved motif and gene duplication provide insights into the evolutionary process for the Hsp70 gene family in cucumber. The promoter analysis showed that the CsHsp70 genes may be involved in the hormone and stress responses of cucumber. Expression analysis by RNA-seq and qRT-PCR suggested that the CsHsp70 genes participate in the regulation of responses to distinct abiotic and biotic stresses. The findings provided by this work will not only offer new insights into the functions of CsHsp70 genes but also help to develop new resistant varieties for cucumber breeding.

Supplementary Materials

The following supporting information can be downloaded at: https://www.mdpi.com/article/10.3390/horticulturae9091057/s1. Table S1: The gene-specific primers used for qRT-PCR. Table S2: Hsp70 proteins from different plant species used in this study. Table S3: Sequences and lengths of motifs among cucumber Hsp70 proteins.

Author Contributions

Data curation, Z.Z., Y.Z. (Yuelong Zhou) and Y.Z. (Yong Zhou); Funding acquisition, H.W.; Investigation, Z.Z. and L.X.; Methodology, Z.Z., L.X. and J.Z.; Project administration, S.L. and H.W.; Resources, J.Z., Z.H., Y.Z. (Yuelong Zhou) and S.L.; Software, L.X.; Supervision, Y.Z. (Yong Zhou); Validation, L.X. and Z.H.; Visualization, S.L.; Writing—original draft, H.W. and Y.Z. (Yong Zhou); Writing—review and editing, Y.Z. (Yong Zhou). All authors have read and agreed to the published version of the manuscript.

Funding

This work was supported by the Open Fund of the Guangdong Provincial Key Laboratory of Utilization and Conservation of Food and Medicinal Resources in Northern Region (FMR2023002M), the Key Projects of Shaoguan University (SZ2019ZK10), and the Science and Technology Planning Project of Shaoguan (210726124532352).

Data Availability Statement

All data are within the manuscript and its Supplementary Files.

Conflicts of Interest

The authors declare no conflict of interest.

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Figure 1. Phylogenetic tree of Hsp70 proteins in cucumber (Cucumis sativus), Arabidopsis (Arabidopsis thaliana), rice (Oryza sativa), pepper (Capsicum annuum), maize (Zea mays), and tomato (Solanum lycopersicum). Four groups (A–D) are represented by different colors, and CsHsp70s are represented by stars. The accession numbers of Hsp70 proteins are provided in Supplementary Table S2.

Figure 2. Conserved motif analysis of CsHsp70 proteins on the basis of the phylogenetic relationship. Ten motifs were marked by different colors and length of box denotes motif length.

Figure 3. Distribution of the CsHsp70 genes on the five chromosomes of cucumber. The bar located on the left side representing the chromosome length was showed in megabase (Mb), and segmental duplication genes are marked with red and connected with red lines.

Figure 4. Phytohormone- and stress-responsive cis-elements in CsHsp70s promoter regions. The amounts of cis-elements in CsHsp70s promoter regions were displayed as different colors and numbers in the grid.

Figure 5. The transcription levels of CsHsp70 genes under salt stress (A), different temperature and photoperiod combined conditions (B) based on RNA-seq data. The red to blue colors on the scale located on the right side representing high to low gene expression, which were calculated as the log2(TPM+1) values. CK-L and Na-L, leaf sample from control and salt-stressed plants; CK-R and Na-R, root sample from control and salt-stressed plants. HL and LL, high and low temperature under long day; HS and LS, high and low temperature under short day.

Figure 6. Transcription levels of five selected CsHsp70 genes under drought (A) and ER stress (B) treatments, as examined using qRT-PCR. Asterisks indicate significant differences examined by the student’s t-test (p < 0.05).

Figure 7. Heat map of differential transcription of CsHsp70 genes under inoculation of PM (Sphaerotheca fuliginea) (A), RKN (Meloidogyne incognita) (B), and DM (Pseudoperonospora cubensis) (C). The transcriptional levels of CsHsp70 genes are displayed as log2 transformed TPM values. Different colors on the scale located on the right side represent differential gene expression, which decrease from red to blue.

Table 1. Identification and characterization of Hsp70 gene family in cucumber.

Gene	Accession No. (v2)	Accession No. (v3)	Chromosome: Location	CDS/bp	Physicochemical Parameters of Proteins
Gene	Accession No. (v2)	Accession No. (v3)	Chromosome: Location	CDS/bp	AA	pI	MW/kDa	GRAVY
CsHsp70-1	Csa2G070310.1	CsaV3_2G009170	Chr2: 5,427,208 .. 5,429,443 (+)	1953	650	5.18	71.27995	−0.422
CsHsp70-2	Csa2G122520.1	CsaV3_2G011210	Chr2: 8,176,614 .. 8,180,834 (+)	2124	707	5.18	75.39607	−0.295
CsHsp70-3	Csa3G147740.1	CsaV3_3G013380	Chr3: 9,928,649 .. 9,930,364 (−)	1716	571	5.52	61.98548	0.040
CsHsp70-4	Csa3G391900.1	CsaV3_3G022450	Chr3: 19,117,817 .. 19,123,998 (−)	2289	762	5.63	85.39136	−0.422
CsHsp70-5	Csa4G179170.1	CsaV3_4G014660	Chr4: 8,965,693 .. 8,969,625 (−)	2043	680	5.69	73.1238	−0.323
CsHsp70-6	Csa4G295440.1	CsaV3_4G024840	Chr4: 11,823,184 .. 11,826,273 (+)	1944	647	5.16	70.86926	−0.403
CsHsp70-7	Csa4G617390.1	CsaV3_4G032260	Chr4: 19,669,174 .. 19,679,176 (+)	2697	898	5.27	100.13606	−0.498
CsHsp70-8	Csa5G149330.1	CsaV3_5G001960	Chr5: 4,469,887 .. 4,473,757 (+)	1998	665	5.10	73.42913	−0.474
CsHsp70-9	Csa5G512930.1	CsaV3_5G026450	Chr5: 17,912,303 .. 17,915,006 (+)	1959	652	5.10	71.45378	−0.442
CsHsp70-10	Csa5G514500.1	CsaV3_5G026520	Chr5: 17,969,849 .. 17,972,430 (−)	1947	648	5.21	71.04657	−0.415
CsHsp70-11	Csa7G312930.1	CsaV3_7G024760	Chr7: 10,868,096 .. 10,872,494 (+)	2001	666	5.09	71.51079	−0.250
CsHsp70-12	Csa7G446710.1	CsaV3_7G033150	Chr7: 17,685,920 .. 17,691,540 (−)	2532	843	5.40	92.89913	−0.430

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Zhou, Z.; Xiao, L.; Zhao, J.; Hu, Z.; Zhou, Y.; Liu, S.; Wu, H.; Zhou, Y. Comprehensive Genomic Analysis and Expression Profile of Hsp70 Gene Family Related to Abiotic and Biotic Stress in Cucumber. Horticulturae 2023, 9, 1057. https://doi.org/10.3390/horticulturae9091057

AMA Style

Zhou Z, Xiao L, Zhao J, Hu Z, Zhou Y, Liu S, Wu H, Zhou Y. Comprehensive Genomic Analysis and Expression Profile of Hsp70 Gene Family Related to Abiotic and Biotic Stress in Cucumber. Horticulturae. 2023; 9(9):1057. https://doi.org/10.3390/horticulturae9091057

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Zhou, Zixian, Lingdi Xiao, Jindong Zhao, Zhaoyang Hu, Yuelong Zhou, Shiqiang Liu, Hao Wu, and Yong Zhou. 2023. "Comprehensive Genomic Analysis and Expression Profile of Hsp70 Gene Family Related to Abiotic and Biotic Stress in Cucumber" Horticulturae 9, no. 9: 1057. https://doi.org/10.3390/horticulturae9091057

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Zhou, Z., Xiao, L., Zhao, J., Hu, Z., Zhou, Y., Liu, S., Wu, H., & Zhou, Y. (2023). Comprehensive Genomic Analysis and Expression Profile of Hsp70 Gene Family Related to Abiotic and Biotic Stress in Cucumber. Horticulturae, 9(9), 1057. https://doi.org/10.3390/horticulturae9091057

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