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Article

Genome-Wide Identification of the Hsp70 Gene Family in Grape and Their Expression Profile during Abiotic Stress

by
Xinna Liu
1,2,
Haiyang Chen
1,2,
Shenchang Li
1,2 and
Lijun Wang
1,*
1
Beijing Key Laboratory of Grape Science and Enology and Key Laboratory of Plant Resources, Institute of Botany, Chinese Academy of Sciences, Beijing 100093, China
2
University of Chinese Academy of Sciences, Beijing 100049, China
*
Author to whom correspondence should be addressed.
Horticulturae 2022, 8(8), 743; https://doi.org/10.3390/horticulturae8080743
Submission received: 18 July 2022 / Revised: 4 August 2022 / Accepted: 15 August 2022 / Published: 18 August 2022
(This article belongs to the Special Issue Stress Biology of Horticultural Plants)
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Abstract

:
Plants encounter a variety of abiotic stresses such as global climate change. Hsp70, as one of the main families of heat shock proteins (Hsps), has a great role in maintenance of the development and growth, and response to abiotic stress. Grape is a very popular fruit worldwide with a high economic value. However, the Hsp70 gene family has not been thoroughly identified in grape (Vitis vinifera L.). In this study, a total of 33 VvHsp70 genes were identified and divided into four clades in V. vinifera. Phylogenetic analysis, gene structure, conserved motif, and duplication events were performed for VvHsp70 genes. The detailed information showed that the VvHsp70 genes clustered together based on the phylogenetic tree had similar subcellular localization, gene structures, and conserved motifs, although there are exceptions. The expression patterns of VvHsp70, VdHsp70, or VaHsp70 were explored in development and abiotic stress including heat, osmotic, and cold stresses by transcription data or qRT-PCR. The results showed that grape Hsp70 genes had strong response for these abiotic stresses, particularly in heat and cold treatments in a different expression pattern. Most of the VdHsp70 genes were upregulated in response to heat treatments while VaHsp70 genes were downregulated in response to cold treatments. Together, our results revealed a new insight for the Hsp70 gene family in grape and will afford fundamental knowledge for further functional analysis and breeding of stress-tolerant grapevines.

1. Introduction

With climate change, plants encounter a variety of abiotic stresses, such as heat or cold temperature, drought, and salinity stress [1]. These abiotic stresses are major environmental factors that limit the productivity of plants. As sessile organisms, plants must evolve regulatory mechanisms to cope with environmental stresses.
Heat shock proteins (Hsps) play an important role in protein folding, assembly, transport, and degradation during plant growth and development. Hsps, as molecular chaperones, play a crucial role in preventing protein denaturation and aggregation during extreme high temperatures [2]. Hsps/chaperone networks have been implicated in the heat stress response, meanwhile they are a major component of a variety of stress responses [3]. Hsps are produced in response to high temperatures or other forms of stress. Swindell et al. (2007) reported that Hsps expression was strongly induced by heat, cold, salt, and osmotic stress [4]. In plants, Hsps are classified into five main families according to their average molecular weight which are Hsp100, Hsp90, Hsp70, Hsp60, and the small Hsp (sHsp) [5]. Although the major Hsps can alleviate problems caused by protein misfolding and aggregation, each major Hsps family has a unique mechanism of action [6].
Hsp70 is a major and highly conserved stress-activated protein in organisms, although several Hsp70 proteins play an important role in housekeeping activities under normal condition [7]. Many studies have shown that there is a relationship between the expression of Hsp70 and abiotic stress in plants. Recently, more Hsp70 genes were reported in response to abiotic stress, such as in Arabidopsis, rice, maize, and tobacco [8,9,10,11]. Hsp70-3 interacted with phospholipase and participated in heat stress defense in Arabidopsis [12]. Overexpression of herbaceous peony Hsp70 improved the thermotolerance of Arabidopsis [13]. A fungal Hsp70 conferred tolerance to heat and other abiotic stresses (osmotic, salt, and oxidative stresses) in Arabidopsis [14]. Overexpression of NtHSP70-1 (a number of Hsp70 in Nicotiana tabacum) improved thermoprotective activity and drought-stress tolerance in plants [15]. Besides the heat response, Hsp70 genes also play an important role in drought and cold tolerance. PtHsp70s contributed to drought tolerance in poplar [16]. Some GmHSP70 genes were upregulated after treatments with PEG which emulates the drought condition [17]. Overexpression of Erianthus arundinaceus Hsp70 improved drought tolerance in sugarcane [18]. The expression of potato StHsp70-1/10/17/20 genes were improved after cold treatments [19]. KoHsp70 improved significantly in transcriptional expression after 48 h cold stress and might play a role in cold stress protective response for Kandelia obovate [20].
Grape (Vitis vinifera L.) is a popular cultivated fruit worldwide, providing table grapes, the basis for wine making, dried fruits, and juices. In recent years, global warming has led to frequent extreme high temperatures, which have seriously affected the yield and quality of fruit trees, including grapes. High temperature often influences the growth and development of grapes, causing reduced quality and production. Besides heat stress, other abiotic stresses (such as drought, cold, and salinity) also affect the distribution and productivity of grapes [21,22]. Hsps are reported to participate in growth, development, and abiotic response in plants [3]. Among them, the Hsp70 family has been identified in several species, such as Arabidopsis, rice, maize, and soybean [8,9,11,17]. Nonetheless, the Hsp70 family in grape has not been studied systematically. What is more, the transcription patterns of grape Hsp70 are lacking during development and abiotic stress.
In this study, we performed a genome-wide analysis of the Hsp70 family in grape based on 12× grape genomes (V. vinifera), and 33 VvHsp70 genes were identified. The phylogeny, gene information, structure, and duplication of VvHsp70 were conducted. The expression profile of VvHsp70 was studied in different tissues and development stages of grape using public transcriptome data. In addition, Vitis davidii Foex is wild species with high thermotolerance while Vitis amurensis Rupr has high tolerance to drought and cold stress [21,23,24,25]. Therefore, transcriptional expression of VdHsp70 (homologs of VvHsp70 in V. davidii) was systematically measured in response to heat. Expression of VaHsp70 (homologs of VvHsp70 in V. amurensis) was systematically measured in osmotic and cold stresses. This study characterized the grape Hsp70 family for the first time and analyzed the expression of grape Hsp70 genes under different abiotic stresses. The results provide fundamental knowledge of Hsp70 in grape and will afford insights for future analysis of the function of Hsp70 in abiotic stress.

2. Materials and Methods

2.1. Identification of Hsp70 Proteins in Grape

Published Arabidopsis and rice Hsp70 family members were used in the BLAST search to obtain candidate genes of the Hsp70 family in grape [8,9]. The grape genome sequence was obtained from the Grape Genome Browser (12×) (http://www.genoscope.cns.fr/externe/GenomeBrowser/Vitis/, accessed on 8 May 2018). Next, a Hidden Markov Model (HMM) was built to search the sequences belonging to the Hsp70 family in grape. The genes obtained from BLAST and HMM were combined. To further determine the reliability of Hsp70 family genes in grape, all candidate non-redundant protein sequences were confirmed with NCBI-Conserved Domain Database (https://www.ncbi.nlm.nih.gov/Structure/cdd/wrpsb.cgi, accessed on 8 January 2020).

2.2. Multiple Alignment and Phylogenetic Analysis

Firstly, the putative Hsp70 family protein sequences of Arabidopsis, rice, and grape were aligned using Clustalx2. Then, the results of alignment were opened by MEGA to build a phylogenetic tree. The Neighbor-Joining method and 1000 bootstrap replications were applied in the building of the phylogenetic tree. Finally, the tree was decorated by EvolView (http://www.evolgenius.info/evolview/, accessed on 13 June 2012). VvHsp70 proteins were named based on their phylogenetic relationships with AtHsp70 [26]. When VvHsp70 members were present in the one-to-one orthology with the Arabidopsis Hsp70 family, the VvHsp70 genes were given the corresponding Arabidopsis-like name (e.g., AtHsp70-5 and VvHsp70-5). The grape genes that had the same phylogenetic distance from a single homologue in Arabidopsis were labeled by a letter (e.g., AtHsp70-17, VvHsp70-17a, VvHsp70-17b, and VvHsp70-17c). As for the remaining VvHsp70 members which were no homologues in Arabidopsis, we named them from VvHsp70-19 to VvHsp70-35 in order according to the adjacent subclass.

2.3. Analysis of Subcellular Localization, Gene Structure, and Conserved Motif

Subcellular localization of VvHsp70 was predicted by Wolf PSORT (https://www.genscript.com/wolf-psort.html, accessed on 21 May 2007) and CELLO v.2.5 (http://cello.life.nctu.edu.tw/, accessed on 21 May 2020, Yu CS, Xinzhu, Taiwan, China). The gene structure of VvHsp70 was determined based on alignment of their coding sequences and respective full-length sequences, meanwhile a diagram of the gene structure was obtained from The Gene Structure Display Server (http://gsds.cbi.pku.edu.cn/, accessed on 10 September 2021). The conserved motif was researched using the MEME program (http://meme-suite.org/tools/meme, accessed on 10 February 1994) according to VvHsp70 amino acid sequences. The motif distribution type was any number of repetitions. The number of motifs just showed the first 10 enrichments.

2.4. Gene Location and Duplication Analysis

Gene location was obtained based on genome annotation files (http://www.grapeworld.cn/ggh/pn.html, accessed on 8 May 2018). The gene location was shown in a circle chromosome map constructed by CIRCOS [27]. The duplication analysis was studied with a multiple collinearity scan toolkit (MCScanX) [28], with the e-value of 1 × 10−5, and blocks containing more than five genes were selected. The duplication events related to VvHsp70 genes were selected and exhibited by CIRCOS, and the connection line linked the putative duplication genes.

2.5. Microarray Data Analysis

For analysis of the expression profiles of grape Hsp70 genes, our previous published transcription data (GSE89113) [29] and public microarray data (PRJNA549981 and GSE36128) [21,30] were used for analysis the grape Hsp70 expression patterns after abiotic stress (heat, osmotic, and cold stress) and during grape development. The transcription data were normalized using SPSS21.0. The pheatmap package of R was used for construction of the expression heatmap.

2.6. Plant Materials and Treatments

V. davidii was used for heat treatment in our study because of its thermotolerance. The grapevine growth condition was performed as described by Xu et al. [23]. The grapevines were grown in the Germplasm Repository for Grapevines in the Institute of Botany of the Chinese Academy of Sciences, located in Beijing, in the spring of 1993. Heat treatments were carried out according to Xu et al. (2014) [23] and Liu et al. (2018) [24]. Heat treatments processes were as follows: the leaves of diameter 5.5 cm were put into a small vessel made of aluminum foil embedded in moist filter paper. The vessel was floated in a temperature-controlled water bath. Leaf discs were treated as follows: (1): 25 °C 2 h and 40 min (Control); (2): 25 °C 2 h and 47 °C 40 min (RT + HS); and (3): 38 °C 2 h and 47 °C 40 min (HA + HS). RT, HA, and HS were abbreviations of room temperature, heat acclimation, and heat stress, respectively. They were sampled after treatments. Each treatment has three biological repeats.
V. amurensis was used in this study for osmotic and cold stress treatments because of its tolerance to osmotic and cold stress. The growth conditions of V. amurensis plantlets were created as described by Xu et al. (2020) [21]. The plantlets were cultured in tissue culture bottles with medium. Similarly, osmotic stress and cold stress treatments were carried out according to Xu et al. (2020) [21]. Six-week-old V. amurensis plantlets in liquid medium supplemented with 8% PEG-6000 (BioRular, Danbury, CT, USA) were used for the osmotic stress treatment, and the same plantlets without PEG-6000 were used as the control. Six-week-old V. amurensis plantlets in solid medium were treated at 4 °C for 2, 4, 8, 12, and 24 h, and the similar plantlets grown at 25 °C were used as the control. Each treatment had three biological replicates; each biological replicate had three plantlets.

2.7. RNA Isolation and Quantitative Real-Time PCR (qRT-PCR) Analysis

RNA of all samples was extracted using the RNApre Pure Plant Kit (Polysaccharides and Polyphenolics-rich) (TIANGEN, Beijing, China). The quality of RNA was detected by agarose gel of 1% concentration. Qualified RNA was reverse transcribed into complementary DNA (cDNA) using HiScript® II Q RT SuperMix for qPCR (+gDNA wiper) (Vazyme, Nanjing, China). qRT-PCR was performed using the AceQ® qPCR SYBR® Green Master Mix (Vazyme, Nanjing, China) and Opticon thermocycler (CFX Connect Real-Time System; Bio-Rad, Hercules, CA, USA). The procedure was 95 °C for 10 min; 40 cycles of 95 °C for 10 s and 60 °C for 30 s; melt curve was analyzed through increments of 0.5 °C for 5 s from 65 to 95 °C. Primers were designed by NCBI Primer-BLAST (https://www.ncbi.nlm.nih.gov/tools/primer-blast/index.cgi?LINK_LOC=BlastHome, accessed on 18 June 2012). VvActin-7 (LOC100232866) was used as an internal control for normalization [31,32,33]. The relative expression level of each gene was calculated using the 2−ΔΔCT method. qRT-PCR was conducted based on three biological replicates and technical replicates. The primers used in this study are listed in Table S1.

3. Results

3.1. Identification and Phylogenetic Analysis of Hsp70 Genes in Grapevine

Thirty-three putative VvHsp70 genes were obtained through Hidden Markov Model (HMM) and BLAST search methods, the Hsp70 proteins of Arabidopsis and rice were referenced [8,9]. Every putative VvHsp70 gene was further identified using NCBI-Conserved Domains (https://www.ncbi.nlm.nih.gov/Structure/cdd/wrpsb.cgi, accessed on 8 January 2020). Finally, 33 VvHsp70 genes were identified in grape.
To determine the phylogenetic relationships among VvHsp70 genes, a phylogenetic analysis of 33 VvHsp70 with 18 Arabidopsis Hsp70 and 32 rice Hsp70 superfamily proteins was built by generating a neighbor-joining phylogenetic tree. The results showed that 33 VvHsp70 were classified into four clades, which is consistent with rice. As shown in Figure 1 and Table 1, Clade I had 17 VvHsp70 genes. However, Clades II, III, and IV contained 5, 3, and 8 VvHsp70 genes, respectively. According to the latest grapevine gene nomenclature system [34], all the VvHsp70 genes were named. The Gene ID corresponding to VvHsp70 gene name is shown in Table 1. The gene lengths of VvHsp70 were different, ranging from 129 bp (VvHsp70-26) to 16,141 bp (VvHsp70-17c). The length of coding sequence (CDS) ranged from 108 bp (VvHsp70-28) to 2805 bp (VvHsp70-17c), corresponding to 35–934 amino acids (Table 1).

3.2. Subcellular Location, Gene Structure, and Conserved Motif Analysis

The VvHsp70 protein subcellular localization prediction was based on the Wolf PSORT and CELLO v.2.5 (http://cello.life.nctu.edu.tw/, accessed on 21 May 2007, Yu CS, Xinzhu, Taiwan, China) software. The predicted results showed that most of VvHsp70 genes in Clade I were cytoplasm and nucleus subcellular localization and one in chloroplast. VvHsp70 genes of Clade II were chloroplast or mitochondria localization. VvHsp70 genes of Clade III were located in the endoplasmic reticulum (ER). Clade IV VvHsp70 genes were cytoplasm, nucleus, ER, and chloroplast localization. Overall, 16 VvHsp70 genes were localized in the cytoplasm, 5 in the chloroplast, 5 in the nuclear, 4 in the endoplasmic reticulum, and 3 in the mitochondria (Table 1).
The gene structure of VvHsp70 was obtained by Gene Structure Display Server 2.0 (http://gsds.cbi.pku.edu.cn/, accessed on 10 September 2021) based on available information from the grape genome annotation file. As shown in Figure 2 and Table 1, the number of exons in all VvHsp70 genes ranged from 1 to 16, whereas the gene structure was conserved in the paralogous pairs from each clade derived from phylogenetic analysis. Most of the VvHsp70 genes in Clade I had 1 or 2 exons; the vast majority of VvHsp70 genes in Clades II and III had 6, 7, or 8 exons; while VvHsp70 genes in Clade IV contained 9 or more (14, 15, or 16) exons in general.
To analyze motif distribution, 10 conserved motifs were revealed in VvHsp70 genes (Figure 3). The motif numbers varied from 0 to 10, and the length of motif ranged from 21 (motif 10) to 50 (motif 1–3, 6, and 7) amino acids (Table S2). Nine VvHsp70 genes had 10 motifs, 4 VvHsp70 genes had 9 motifs, 2 VvHsp70 genes had 6 motifs, 3 VvHsp70 genes had 5 motifs, 1 VvHsp70 gene had 3 motifs, 2 VvHsp70 genes had 2 motifs, 5 VvHsp70 genes had only 1 motif, and no common motif was found in 7 VvHsp70 genes.

3.3. Gene Location and Gene Duplication

A total of 33 VvHsp70 genes distributed in 16 chromosomes except for chromosomes 1, 10, and 12 (Figure 4 and Table 1). Among them, chromosomes 2 and 7 had the highest members, with 3 VvHsp70 genes; chromosomes 4, 6, 9, 13, 16, 17, 18, and 19 had the same members, with 2 VvHsp70 genes; chromosomes 3, 5, 8, 11, 14, and 15 had only 1 VvHsp70 gene; the location of VvHsp70-9/22/29/30/32 were unknown (Figure 4 and Table 1). In addition, we investigated the gene duplication events of the VvHsp70 genes. At least 16 segmental duplication events were identified, including VvHsp70-12/VvHsp70-11, VvHsp70-10/VvHsp70-17a, VvHsp70-17b/VvHsp70-21, VvHsp70-8/VvHsp70-20, VvHsp70-20/VvHsp70-22, VvHsp70-20/VvHsp70-26, VvHsp70-20/VvHsp70-23, VvHsp70-31/VvHsp70-9, VvHsp70-27/VvHsp70-7, VvHsp70-21/VvHsp70-24, VvHsp70-14/VvHsp70-6, VvHsp70-17c/VvHsp70-7, VvHsp70-5/VvHsp70-9, VvHsp70-6/VvHsp70-35, and VvHsp70-22/VvHsp70-9 (Figure 4). There were no tandem duplication events between VvHsp70 genes.

3.4. Expression Patterns of VvHsp70 Genes in Different Tissues and Development Stages

To explore the temporal and tissue-specific expression of VvHsp70 in grapevine, a heatmap of VvHsp70 gene transcription patterns was performed using the published grapevine microarray data GSE36128 [30]. As shown in Figure 5, different VvHsp70 gene expression levels varied greatly. The detailed information about the sample is listed in Table S3. VvHsp70-32, VvHsp70-23, and VvHsp70-12 were expressed abundantly in various tissue and developmental stages, while a large number of VvHsp70 genes were expressed in very low levels such as VvHsp70-25, VvHsp70-26, and so on. VvHsp70-30 and VvHsp70-5 were expressed strongly in Leaf-s (senescing leaf) and Bud-w (winter bud), respectively. In addition, we conducted a principal component analysis (PCA) based on these microarray data. The results showed that these VvHsp70 genes did not show obvious tissue/organ specificity (Figure S1).

3.5. Expression Patterns of Grape Hsp70 Genes in Response to High Temperatures

We investigated the response of VvHsp70 genes to heat stress using our previously published RNA-seq data GSE89113. The expression of 20 VvHsp70 genes is shown in a heatmap excluding 13 genes in which their expression in RNA-seq data was not detected. The results showed that the expression levels of 18 VvHsp70 genes were increased after heat stress except for VvHsp70-30 and VvHsp70-16 (Figure 6a).
To further evaluate the function of VdHsp70 genes in basal and acquired thermotolerance, all VdHsp70 genes were analyzed by qRT-PCR after heat treatments in V. daviddi. The results are shown in Figure 6b. In total, 19 VdHsp70 genes were shown, except for 14 VdHsp70 genes whose transcriptional level was not detected or there was no significant difference before and after heat treatments. Almost all VdHsp70 genes’ expressions were upregulated after HA + HS (except for VdHsp70-16). To further analyze the expression of VdHsp70 genes, we firstly compared the transcription expression level of VdHsp70 genes in the treatment of HA + HS with RT + HS, and defined upregulated, unchanged, and downregulated as Groups I, II, and III, respectively. Then, based on the compared RT + HS with control, each group was further classified as sub-groups i (upregulated), ii (unchanged), and iii (downregulated), respectively. As shown in Figure S2, 19 VdHsp70 genes were classified into two groups and four sub-groups. In total, 14 and 5 VdHsp70 genes belong to Groups I and II, respectively. Seven VdHsp70 genes (VdHsp70-5, VdHsp70-8, VdHsp70-10, VdHsp70-12, VdHsp70-19, VdHsp70-20, and VdHsp70-21) belong to Group I-i in which the transcriptional level was upregulated in the treatments of RT + HS and HA + HS, higher in the treatment of HA + HS. Seven VdHsp70 genes (VdHsp70-6, VdHsp70-7, VdHsp70-9, VdHsp70-11, VdHsp70-17a, VdHsp70-22, and VdHsp70-33) belong to Group I-ii in which the transcriptional level was upregulated just in the treatment of HA + HS. Four VdHsp70 genes (VdHsp70-13, VdHsp70-23, VdHsp70-24, and VdHsp70-32) belong to Group II-i in which the transcriptional level was upregulated in the treatments of RT + HS and HA + HS with no significance between both treatments. The VdHsp70-16 (belong to Group II-iii) transcriptional level was downregulated after the treatments of RT + HS and HA + HS with no significance between both treatments.

3.6. VaHsp70 Genes in Response to Osmotic and Cold Stresses

Heat shock proteins are produced in response to high temperatures or other stresses. In order to explore the putative functions of VaHsp70 genes under osmotic and cold stresses, we analyzed the response of VaHsp70 genes to osmotic and cold stresses using RNA-Seq data PRJNA549981 [21]. The results cannot reflect VaHsp70 gene responses to osmotic and cold stresses well (Figure 7a). This may be because there was just one treatment for stress. Therefore, we analyzed their expression level using qRT-PCR after osmotic or cold treatments in V. amurensis systematically. Most of the VaHsp70 genes were not detected after osmotic stress (data not shown). The expression of six VaHsp70 genes changed significantly (Figure 7b). The expression levels of three VaHsp70 genes (VaHsp70-23/29/37) were upregulated after osmotic treatments for 4 or 8 h (Figure 7b). The expression levels of three VaHsp70 genes (VaHsp70-21/32/33) were downregulated after osmotic treatments.
After cold treatments, there was just VaHsp70-19 genes whose expression levels were continuously upregulated under cold stress compared with control (Figure 7c). The expression levels of VaHsp70-13 and VaHsp70-32 were just upregulated after treatments at 4 °C for 24 h. Five VaHsp70 genes (VaHsp70-9/12/17a/21/23) were downregulated after cold treatments for 2/4/8/12 h and returned to the initial level after cold treatment for 24 h. The expression of VaHsp70-7 was downregulated after cold treatments for 2/4 h and recovered to the initial level after cold treatment for 8/12/24 h. As shown in Figure 7c, the other remaining 12 VaHsp70 genes were downregulated in expression level after cold treatments. In addition, we conducted a PCA based on our qRT-PCR for correlation among these grape Hsp70 genes under abiotic stress. The results showed that grape Hsp70 genes were more related with heat stress (Figure S3).

4. Discussion

Hsps play a vital role in the maintenance of homeostasis and response to stress [35]. In grapes, the families of Hsp90, DnaJ (Hsp40), and sHsp have been identified [36,37,38]. When plants are exposed to environmental stresses, Hsp70 protects cells from being damaged. Research on the Hsp70 family in grape is lacking. In this study, we identified and characterized the Hsp70 genes family in grape and explored their expression patterns in abiotic stress.
There were 33 Hsp70 genes in grape, a number higher than Arabidopsis (17 Hsp70) and near to rice (32 Hsp70) [8,9]. Phylogenetic analysis showed that the VvHsp70 genes were divided into four clades based on their amino acids with Arabidopsis and rice. The results were similar with rice which were also classified into four lineages [9]. The gene subcellular location, structure, and motif of VvHsp70 supported further information for their phylogeny. Different subcellular localizations may imply functional specificity and phylogenetic differences [8]. In rice, Hsp70 genes of Clades A and B in in silico localization are nucleus/cytoplasm and ER, respectively. Similarly, the predicted results showed that VvHsp70 genes in Clades I and III were localized in the nucleus/cytoplasm (except for VvHsp70-27 in chloroplast) and ER, respectively. Mitochondria and chloroplast were clustered on Clade C in rice Hsp70. Localization of VvHsp70 genes in Clade II were mitochondria or chloroplast (Table 1) [9]. The gene structure was more similar in the same clade of VvHsp70 (Figure 2). For example, most VvHsp70 genes in Clade I had one or two exons. Clade III VvHsp70 genes had seven or eight exons. Gene structure determined its function in some extent. For instance, the highly similar pairs VvHsp70-9 and VvHsp70-10 located in mitochondria, VvHsp70-11 and VvHsp70-12 located in ER, have the conserved gene structure. This finding can also be observed in other species, such as Arabidopsis, soybean, and rice. In Arabidopsis, AtHsp70 genes with the similar exon–intron organization tend to cluster together, and localized to the same cell compartment [8]. In soybean, the closest GmHsp70 genes of the same subfamily had similar exon/intron structure and intron number [17]. A total of 10 were set to search the consensus motif in VvHsp70. In Figure 3, Clade III and most Clade I VvHsp70 genes contained 10 motifs. Most of Clade II VvHsp70 genes included nine motifs. The motif number varies among Clade IV VvHsp70 genes. The results showed that the members of motif in the VvHsp70 sub-group are relatively conserved except for Clade IV VvHsp70 genes. In potato, the same sub-family of StHsp70 had the similar type, order, and number of motifs, but different from other subfamily proteins [19]. The number and distribution of conserved motifs may be related to the diversity of their gene functions.
Gene duplication is an important method of gene family expansion, which also increases genetic novelty in plant evolutionary events [39]. In this study, we explored the gene duplication events. There were 16 gene duplication events which belong to segmental duplication without tandem duplication. Compared with tandem duplication, there is more segment duplication in the gene family. This is consistent with previous studies in LEA, HSF, DnaJ, and UDP-Glycosyltransferase families of grape [21,24,37,40]. Gene duplication increased the number of gene families. At the same time, function loss due to genetic or chromosomal mutation may be alleviated by gene duplication [10].
Some Hsps are involved in organism development during the absence of stress, although Hsps are firstly characterized because Hsps expression improved after heat treatments [41]. In our study, VvHsp70-12, VvHsp70-23, and VvHsp70-32 expressed higher compared with other VvHsp70 genes during the process of grape development (Figure 5). This result indicated that VvHsp70-12, VvHsp70-23, and VvHsp70-32 may play vital roles in the development of grapes. VvHsp70-5 and VvHsp70-30 showed specific tissue expression (VvHsp70-5 in Bud-W and VvHsp70-30 in Leaf-S). There were studies that showed that HSP/chaperone play an essential role in the proper function of a cell [3]. Hsps, including Hsp70, are important molecular chaperons that maintain cellular balance under both normal and stressful conditions [42]. VvHsp70-5 highly expressed in winter bud, and VvHsp70-30 highly expressed in senescing leaves. They may play a role in maintenance of cellular protein homeostasis in stressed conditions.
Hsp70 is the most abundant heat shock protein that protects plant cells from negative effects of heat stress [7]. Most of VvHsp70 genes were improved after heat treatments except several VvHsp70 genes which were not detected (Figure 6a). Furthermore, we studied the VdHsp70 roles in basal and acquired thermotolerance by qRT-PCR (Figure 6b and Figure S2). Nineteen VdHsp70 genes were classified into four sub-groups based their expression before and after heat treatments. The expression level of Group I-i members was upregulated in treatments with RT + HS and HA + HS, higher in treatments HA + HS. These Group I-i VdHsp70 genes may play an important role in basal and acquired thermotolerance of grape. There was a report that overexpression of the Trichoderma harzianum Hsp70 gene increased Arabidopsis basal and acquired thermotolerance [14]. The expression level of Group I-ii members was upregulated just in treatments of HA + HS. This means that these members may just play a crucial role in acquired thermotolerance of grape. In Arabidopsis, transgenic Hsp70 antisense gene plants had a negative effect on the acquired thermotolerance, while its basal thermotolerance was not affected [43]. What is more, Hsp70-Hsp90 organizing protein played a major role in long-term acquired thermotolerance in Arabidopsis [44]. The expression level of Group II-i genes was upregulated in treatments with RT + HS and HA + HS, with no significant difference between treatments RT + HS and HA + HS. These Group II-i genes may just play a vital role in basal thermotolerance of grapes. Transgenic tobacco seedlings with the NtHSP70-1 in-sense and anti-sense orientations improved and reduced their basal thermotolerance, respectively [15]. Overall, Hsp70 genes play roles in basal and acquired thermotolerance in grapevine with division and cooperation among the Hsp70 gene family.
Drought stress can reduce productivity of plants. Studies have shown that Hsp70 can improve the tolerance of plants to drought stress [7,45]. In tobacco, several NtHsp70 genes were induced by PEG treatments [10]. Expression of 12 HvHsp70 genes was induced in barley after drought stress [46]. PtHsp70s genes showed different expression patterns after drought stress in drought sensitive and resistant Populus species [16]. Since gene expression can provide important clues for gene function, we explored the expression of VaHsp70 in response to osmotic stress in Vitis amurensis. The expression of six VaHsp70s were significantly changed under osmotic stress, and with different change tendencies (Figure 7b). The expression of VaHsp70-23/29/37 was upregulated after osmotic stress for 4 or 8 h, and then returned to a normal or low expression level. The expression of VaHsp70-21/32/33 was downregulated after osmotic stress. These results indicated that different Hsp70 genes may have a diverse regulatory mechanism in response to osmotic stress in grapevine. What is more, more Hsp70 genes were involved in heat stress compared with drought in grape. These results suggested that Hsp70 plays a more important role in heat stress than in osmotic stress in grape.
Several studies reported the role of Hsp70 in imparting tolerance against low temperature stress, such as in wheat, tobacco, and barley [42,47,48,49]. In our study, most of the VaHsp70 genes were changed significantly in response to cold stress by qRT-PCR. VaHsp70-19 hold a high expression level after cold stress while most of the VaHsp70 genes were downregulated in response to cold treatments (Figure 7c). The results implied that VaHsp70 genes may be involved in cold stress through different mechanisms compared with heat. Arabidopsis and Chlamydomonas reinhardtii cells showed increased expression levels of Hsp70 and its chaperones (e.g., Hsp90) after exposure to cold stress [50,51], which reflects an Hsp70 mechanism in respond to cold stress. In Arabidopsis, Hsp70-16 negatively regulated seed germination under cold stress which may be another mechanism to protect plants from cold injury [52]. In a nutshell, Hsp70 genes can be involved in cold response through their up- or downregulation. The mechanism of VaHsp70 in response to cold stress needs to be further explored.
Haider et al. (2017) reported that the two VvHsp70 genes were involved in drought stress in V. vinifera in which one VvHsp70 gene was upregulated and another VvHsp70 gene was downregulated [53]. Compared with the above results, in our study there were more VaHsp70 genes involved in drought stress in V. amurensis than in V. vinifera. There were no Hsp70 genes among the significantly different expressed genes between V. vinifera and V. amurensis after cold stress [54] while most VaHsp70 genes were downregulated after cold treatments in our study. Therefore, whether more Hsp70 genes were involved in drought or cold response in V. amurensis than V. vinifera should be further studied in the same drought or cold stress conditions in the future.
Throughout our research, there were some grape Hsp70 genes such as Hsp70-25/26/27, which had short CDSs in gene structure, and were not shown for motif analysis. What is more, they showed no change in expression level, which means they were not detected in the transcriptome and qRT-PCR. Whether these genes have functions and what functions these genes have need to be further studied.

5. Conclusions

In this study, a total of 33 VvHsp70 genes were identified in grape, and these genes were divided into four clades. Then, a phylogenetic tree, subcellular location, gene structure, conserved motif, and duplication were further analyzed to explore functional characteristics of VvHsp70. In addition, transcriptome data were used to study their expression patterns during different tissue and development stages of grape. Particularly, the expression level of grape Hsp70 genes were examined in response to heat, osmotic, and cold abiotic stress. The results showed that grape Hsp70 genes were more involved in heat and cold stress responses than drought, particularly in heat stress. This study provided novel insights into the response of grape Hsp70 genes to abiotic stress and will have great implications for further research on grape breeding.

Supplementary Materials

The following are available online at https://www.mdpi.com/article/10.3390/horticulturae8080743/s1, Figure S1: Principal component analysis (PCA) for correlation among VvHsp70 genes in different tissues; Figure S2: Expression of VdHsp70 in V. davidii grapevine leaves in response to high temperatures; Figure S3: Principal component analysis (PCA) for correlation among grape Hsp70 genes in different abiotic stresses; Table S1: Sequences of primers used for qRT-PCR in this study; Table S2: Motif information identified by MEME tools in VvHsp70s; Table S3: Description of samples from published data GSE36128.

Author Contributions

Conceptualization, L.W.; methodology, X.L.; software, X.L.; validation, X.L. and L.W.; formal analysis, X.L.; investigation, H.C. and S.L.; resources, L.W.; data curation, X.L.; writing—original draft preparation, X.L.; writing—review and editing, L.W.; visualization, X.L.; supervision, L.W.; project administration, L.W.; funding acquisition, L.W. All authors have read and agreed to the published version of the manuscript.

Funding

This work was supported by the National Key Research and Development Program of China (Grant no. 2018YFD1000300), the National Natural Science Foundation of China (Grant no. 31872065 and U21A20227), and Strategic Priority Research Program of the Chinese Academy of Sciences (Grant No. XDA23080602).

Institutional Review Board Statement

Not applicable.

Informed Consent Statement

Not applicable.

Data Availability Statement

All data in this study can be found in the manuscript or Supplemental Materials.

Conflicts of Interest

The authors declare no conflict of interest.

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Figure 1. Phylogenetic analysis of VvHsp70 proteins with Arabidopsis and rice. Phylogenetic tree of Hsp70 was generated using ClustalW in MEGA based on Hsp70 amino acids of grape, Arabidopsis, and rice. Clades I–IV stand for the subfamilies I to IV.
Figure 1. Phylogenetic analysis of VvHsp70 proteins with Arabidopsis and rice. Phylogenetic tree of Hsp70 was generated using ClustalW in MEGA based on Hsp70 amino acids of grape, Arabidopsis, and rice. Clades I–IV stand for the subfamilies I to IV.
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Figure 2. Gene structure of VvHsp70 genes. The red blocks represent the coding sequence (CDS), the blue blocks represent untranslated region (UTR), and the black lines represent introns. The exons and introns can be estimated using the scale at the bottom. Clades I–IV stand for the subfamilies I to IV.
Figure 2. Gene structure of VvHsp70 genes. The red blocks represent the coding sequence (CDS), the blue blocks represent untranslated region (UTR), and the black lines represent introns. The exons and introns can be estimated using the scale at the bottom. Clades I–IV stand for the subfamilies I to IV.
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Figure 3. The conserved motifs analysis of VvHsp70 genes. The distribution of conserved motifs is shown in (a). The VvHsp70 genes without common conserved motifs are not shown. Different motifs are presents in different colored boxes. The corresponding sequence of the identified motif is shown in (b). Clades I–IV stand for the subfamilies I to IV.
Figure 3. The conserved motifs analysis of VvHsp70 genes. The distribution of conserved motifs is shown in (a). The VvHsp70 genes without common conserved motifs are not shown. Different motifs are presents in different colored boxes. The corresponding sequence of the identified motif is shown in (b). Clades I–IV stand for the subfamilies I to IV.
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Figure 4. Chromosome distribution and duplication analysis of VvHsp70 genes. Different chromosomes are drawn in different colors. The approximate positions of VvHsp70 genes are indicated by short black lines on the circle. Red curves represent the duplication events between VvHsp70 genes. Chrs 1-Un: Chromosome 1-Un.
Figure 4. Chromosome distribution and duplication analysis of VvHsp70 genes. Different chromosomes are drawn in different colors. The approximate positions of VvHsp70 genes are indicated by short black lines on the circle. Red curves represent the duplication events between VvHsp70 genes. Chrs 1-Un: Chromosome 1-Un.
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Figure 5. Expression analysis of VvHsp70 genes in different tissues and development stages based on GSE36128. The color scale represents relative expression level. Detailed tissue information is described in Table S3 [30].
Figure 5. Expression analysis of VvHsp70 genes in different tissues and development stages based on GSE36128. The color scale represents relative expression level. Detailed tissue information is described in Table S3 [30].
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Figure 6. Expression analysis of grape Hsp70 genes in response to heat stress. (a) Expression analysis of VvHsp70 genes in response to heat stress. Transcription data came from our previous research [29]. Some VvHsp70 genes were not shown as their expression was not detected. T25, T35, T40, and T45 mean the expression of VvHsp70 genes after 25, 35, 40, and 45 °C for 2 h. The color scale represents relative expression level. (b) Expression analysis of VdHsp70 genes in response to heat stress by qRT-PCR. V. davidii grape leaves treated with (1): 25 °C 2 h and 40 min, (2): 25 °C 2 h and 47 °C 40 min, and (3): 38 °C 2 h and 47 °C 40 min were marked as Control, RT + HS, and HA + HS, respectively.
Figure 6. Expression analysis of grape Hsp70 genes in response to heat stress. (a) Expression analysis of VvHsp70 genes in response to heat stress. Transcription data came from our previous research [29]. Some VvHsp70 genes were not shown as their expression was not detected. T25, T35, T40, and T45 mean the expression of VvHsp70 genes after 25, 35, 40, and 45 °C for 2 h. The color scale represents relative expression level. (b) Expression analysis of VdHsp70 genes in response to heat stress by qRT-PCR. V. davidii grape leaves treated with (1): 25 °C 2 h and 40 min, (2): 25 °C 2 h and 47 °C 40 min, and (3): 38 °C 2 h and 47 °C 40 min were marked as Control, RT + HS, and HA + HS, respectively.
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Figure 7. Expression of VaHsp70 leaves in response to osmotic and cold stress in V. amurensis. (a) Transcription data came from public transcriptome data PRJNA549981. A, expression of VaHsp70 under no osmotic stress. B, expression of VaHsp70 under osmotic stress for 12 h. C, expression of VaHsp70 under no cold stress. D, expression of VaHsp70 under cold stress for 12 h. (b) Expression of VaHsp70 in response to osmotic treatments. Horizontal axis means hours after osmotic treatments by qRT-PCR. (c) Expression of VaHsp70 in response to cold temperatures by qRT-PCR. Horizontal axis means hours after cold treatments.
Figure 7. Expression of VaHsp70 leaves in response to osmotic and cold stress in V. amurensis. (a) Transcription data came from public transcriptome data PRJNA549981. A, expression of VaHsp70 under no osmotic stress. B, expression of VaHsp70 under osmotic stress for 12 h. C, expression of VaHsp70 under no cold stress. D, expression of VaHsp70 under cold stress for 12 h. (b) Expression of VaHsp70 in response to osmotic treatments. Horizontal axis means hours after osmotic treatments by qRT-PCR. (c) Expression of VaHsp70 in response to cold temperatures by qRT-PCR. Horizontal axis means hours after cold treatments.
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Table
Table 1. Information of VvHsp70 identified in grape.
Table 1. Information of VvHsp70 identified in grape.
Gene Locus IDGene SymbolExonIntronLocalizationCladeChrStart SiteEnd SiteGene Length
(bp)		CDS
(bp)		Protein
(aa)		Strand
VIT_06s0004g04510VvHsp70-1921CytoI65470302547346031591947648−
VIT_06s0004g04470VvHsp70-2021CytoⅠ65418191542067724871959652+
VIT_08s0007g00130VvHsp70-2121CytoⅠ8145185341452157530421950649+
VIT_00s0787g00020VvHsp70-2232ChloⅠUn35359691353611181428831276+
VIT_13s0019g01430VvHsp70-2321CytoⅠ133014601301733227321953650+
VIT_11s0037g00510VvHsp70-510CytoⅠ118347523834965521331971656−
VIT_13s0019g00930VvHsp70-2421CytoⅠ132720364272371633531995664+
VIT_15s0046g02740VvHsp70-2510CytoⅠ151948855219488854303303100−
VIT_17s0000g09950VvHsp70-2610CytoⅠ17119273651192749312912942−
VIT_07s0095g00430VvHsp70-2721ChloⅠ712876279128793963118315104−
VIT_07s0005g06130VvHsp70-2810NuclⅠ7109277941092817237910835+
VIT_00s0198g00100VvHsp70-2910NuclⅠUn109091281090957544826186−
VIT_00s0254g00030VvHsp70-3010CytoⅠUn180002701800121194219564−
VIT_07s0005g06650VvHsp70-3121CytoⅠ712031939120329791041363120−
VIT_00s0254g00040VvHsp70-3210CytoⅠUn180012121800142921819564+
VIT_16s0115g00060VvHsp70-3333NuclⅠ163576063357681074822574−
VIT_02s0012g01830VvHsp70-3410NuclⅠ282600588260480423405134−
VIT_18s0041g01230VvHsp70-687ChloⅡ18258472162585353063152073690+
VIT_17s0000g03310VvHsp70-787ChloⅡ173189808319435545482124707+
VIT_00s0415g00030VvHsp70-965MitoⅡUn282906802829539247132049682+
VIT_03s0097g00530VvHsp70-1065MitoⅡ3108997951090477249782040679+
VIT_18s0075g00260VvHsp70-3521MitoⅡ182162273321623656924408135−
VIT_16s0098g01580VvHsp70-1187ERⅢ16217387592174284040822004667−
VIT_02s0025g02140VvHsp70-1287ERⅢ21900784190480640232004667+
VIT_14s0060g02340VvHsp70-1376ERⅢ141920620192424136221950649+
VIT_05s0020g03330VvHsp70-811CytoⅣ55092723509621634941740579+
VIT_09s0002g04680VvHsp70-1498CytoⅣ94243222424888856672547848+
VIT_19s0090g00340VvHsp70-1698NuclⅣ196484998649560210,6052316771+
VIT_19s0014g03420VvHsp70-3643CytoⅣ193567945357397660321257418−
VIT_02s0012g00960VvHsp70-3732CytoⅣ26881520688284813291149382+
VIT_04s0043g00710VvHsp70-17a1513CytoⅣ4146921951470514112,9472223740−
VIT_04s0008g04410VvHsp70-17b1413ERⅣ43800929381202611,0982688895−
VIT_09s0096g00090VvHsp70-17c1615ChloⅣ9114860351150217516,1412805934+
Note: Cyto, Nucl, Chlo, Mito, and ER are abbreviations of cytoplasm, nucleus, chloroplast, mitochondria, and endoplasmic reticulum, respectively. “Un” means that location of the gene was unknown. “+” and “−“ mean the gene translate in forward and reverse direction, respectively.
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MDPI and ACS Style

Liu, X.; Chen, H.; Li, S.; Wang, L. Genome-Wide Identification of the Hsp70 Gene Family in Grape and Their Expression Profile during Abiotic Stress. Horticulturae 2022, 8, 743. https://doi.org/10.3390/horticulturae8080743

AMA Style

Liu X, Chen H, Li S, Wang L. Genome-Wide Identification of the Hsp70 Gene Family in Grape and Their Expression Profile during Abiotic Stress. Horticulturae. 2022; 8(8):743. https://doi.org/10.3390/horticulturae8080743

Chicago/Turabian Style

Liu, Xinna, Haiyang Chen, Shenchang Li, and Lijun Wang. 2022. "Genome-Wide Identification of the Hsp70 Gene Family in Grape and Their Expression Profile during Abiotic Stress" Horticulturae 8, no. 8: 743. https://doi.org/10.3390/horticulturae8080743

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