Next Article in Journal
Optimized Prime Editing of Human Induced Pluripotent Stem Cells to Efficiently Generate Isogenic Models of Mendelian Diseases
Next Article in Special Issue
Comprehensive Identification of AREB Gene Family in Populus euphratica Oliv. and Functional Analysis of PeAREB04 in Drought Tolerance
Previous Article in Journal
microRNA as an Important Mediator in the Regulation of Male Gallus gallus domesticus Reproduction: Current State of the Problem
Previous Article in Special Issue
Regulatory Mechanism of Exogenous ABA on Gibberellin Signaling and Antioxidant Responses in Rhododendron chrysanthum Pall. Under UV-B Stress
 
 
Search for Articles:
Title / Keyword
Author / Affiliation / Email
Journal
Article Type
 
 
Advanced Search
 
Section
Special Issue
Volume
Issue
Number
Page
 
Journals
IJMS
Volume 26
Issue 1
10.3390/ijms26010102
ijms-logo
Submit to this Journal Review for this Journal Propose a Special Issue
► Article Menu

Article Menu

share Share announcement Help format_quote Cite question_answer Discuss in SciProfiles
first_page
settings
Order Article Reprints
Font Type:
Arial Georgia Verdana
Font Size:
Aa Aa Aa
Line Spacing:
Column Width:
Background:
Article

Genome-Wide Identification and Expression Analysis of HSP70 Gene Family Under High-Temperature Stress in Lettuce (Lactuca sativa L.)

by
Qian Wang
1,†,
Wenjing Sun
1,†,
Yipei Duan
1,
Yikun Xu
2,
Huiyu Wang
1,
Jinghong Hao
1,
Yingyan Han
1 and
Chaojie Liu
1,*
1
College of Plant Science and Technology, Beijing University of Agriculture, Beijing 102206, China
2
College of Food Science and Engineering, Shandong Agricultural University, Taian 271018, China
*
Author to whom correspondence should be addressed.
†
These authors contributed equally to this work.
Int. J. Mol. Sci. 2025, 26(1), 102; https://doi.org/10.3390/ijms26010102
Submission received: 19 November 2024 / Revised: 17 December 2024 / Accepted: 19 December 2024 / Published: 26 December 2024
(This article belongs to the Special Issue Transcriptional Regulation in Plant Development: 2nd Edition)
Browse Figures
Review Reports Versions Notes

Abstract

:
The heat shock protein 70 (HSP70) family plays an important role in the growth and development of lettuce and in the defense response to high-temperature stress; however, its bioinformatics analysis in lettuce has been extremely limited. Genome-wide bioinformatics analysis methods such as chromosome location, phylogenetic relationships, gene structure, collinearity analysis, and promoter analysis were performed in the LsHSP70 gene family, and the expression patterns in response to high-temperature stress were analyzed. The mechanism of LsHSP70-19 in heat resistance in lettuce was studied by virus-induced gene silencing (VIGS) and transient overexpression techniques. The results showed that a total of 37 LsHSP70 genes were identified by the Hidden Markov Model (HMM) and Protein Family Database (Pfam). These 37 LsHSP70 genes were classified into groups A, B, C, and D by phylogenetic relationships. They were mainly localized on seven chromosomes except for chromosome 3; gene structure analysis showed that LsHSP70 contained 1–9 exons, and the protein structure domains of genes in the same group were highly conserved. The covariance analysis showed that nine pairs of LsHSP70 genes existed between LsHSP70 members, and lettuce LsHSP70 and sunflower HaHSP70 had been more conserved in the evolutionary process. The promoter analysis showed that there were a large number of cis-acting elements related to phytohormones, growth, development, stress, and light response in LsHSP70. In addition, the results of the expression pattern analysis for all LsHSP70 genes under high-temperature stress showed that 28 out of 37 LsHSP70 genes were able to respond to heat stress, and only LsHSP70-8, LsHSP70-14, LsHSP70-19, LsHSP70-23, and LsHSP70-24 were able to respond rapidly to heat stress (2 h). The expression of LsHSP70-19 was higher at different periods under high-temperature stress; the overexpression of LsHSP70-19, the plant fresh weight, and the root weight were better than the control (CK); and the heat resistance was better. These results suggest that LsHSP70-19 may play an important role under high-temperature stress in lettuce.

1. Introduction

High temperature is one of the unfavorable environmental factors in crop growth and development, and high-temperature stress can cause irreversible damage to plants when the ambient temperature is persistently higher than the optimum temperature for plant growth. Heat shock proteins (HSPs), produced as a result of high-temperature stress, are present in almost all organisms and are highly conserved, with low levels under normal conditions, but they are synthesized in large quantities in a short period of time when stimulated by unfavorable conditions in the external environment [1]. HSPs can be categorized as HSP100, HSP90, HSP70, HSP60, and small-molecule heat shock proteins (sHSPs) based on their molecular weight [2]. HSPs mainly act as molecular chaperones during plant growth and development to assist in various types of protein folding, ensure the correct folding of proteins, avoid damage to proteins, maintain the stability of the plant internal environment, and play an important role in plant resistance to adversity stress [3].
HSP70 is usually categorized as HSP70, HSC70 (heat shock cognate 70), mtHSP70, or BiP/Grp78 (binding immunoglobulin protein) [4]. The HSP70 gene family generally has two typically conserved structural domains. Genes are enriched on chromosome 9. The C-terminal end consists of two subregions, the SBD and the C-terminal “lid”, which assists in the substrate binding of the SBD, and it binds, recognizes, and processes substrate proteins, while the NBD at the N-terminal end is a highly conserved region, which binds and hydrolyzes ATP [5,6].
HSP70 plays an important role in plant growth, development, and resistance to various stresses [7]. Under various biotic and abiotic stresses, the expression level is often higher, and the anti-stress ability of plants is improved. The chili CaHSP70-2 gene was overexpressed in Arabidopsis thaliana to enhance its tolerance to high temperature [8]. The overexpression of Arabidopsis cytoplasmic HSC70-1 effectively enhanced heat tolerance in transgenic plants [9]. The overexpression of tobacco NtHSP70-1 elevated plant tolerance to drought stress [10]. The expression of TaHSC70 in the leaves of yellow-flowering seedlings was increased after heat stress in wheat seedlings at 40 °C [11]. It was shown that when the thermosensitive mutant carrying the T-DNA insertion HSP70 gene OsHSP70CP1 grew at the high temperature of 40 °C, the seedling leaves showed severe yellowing, and the plant phenotype was normal at 27 °C [12]. In addition, HSP70 is involved in the high-temperature stress response in plants such as grape and chrysanthemum [13,14]. Currently, the involvement of HSP70 under high-temperature stress has been reported in Arabidopsis (Arabidopsis thaliana L.) [15], spinach (Spinacia oleracea L.) [16], chili pepper (Capsicum annuum L.) [8], wheat (Triticum aestivum L.) [11], rice (Oryza sativa L.) [17], potato (Solanum tuberosum L.) [7], soybean (Glycine max L.) [18], and cotton (Gossypium hirsutum L.) [19] regarding the identification and expression analysis of the HSP70 gene family.
Lettuce is a leafy vegetable, widely grown around the world, that prefers coolness and does not tolerate high temperatures [20]. It is rich in nutrients and minerals. Like some Asteraceae crops, it is susceptible to high temperatures during growth and development with lower quality and yield [14]. The identification of the HSP70 gene family in lettuce and its functional validation have not yet been reported. The physicochemical properties, phylogenetic relationships, gene structure, and role elements in the promoter region of LsHSP70 were analyzed. The expression pattern of LsHSP70 under heat stress for 2 h and 4 days were studied, the LsHSP70-19 genes that responded rapidly to heat stress were screened out, and we initially verified the positive effect on lettuce heat tolerance by VIGS and transient overexpression techniques. The positive effect on heat tolerance in lettuce was initially verified. A preliminary framework for exploring lettuce HSP70 genes was constructed. We provide a theoretical basis for the subsequent analysis of the role of LsHSP70 lettuce under high-temperature stress.

2. Results

2.1. Identification, Chromosomal Localization and Physicochemical Properties of HSP70 Family Members in Lettuce

Based on the structural sequence of Arabidopsis HSP70, 37 HSP70 genes in lettuce were mined. The conserved structural domains of all the putative 37 genes by Pfam and CDD databases were identified. Eventually, 37 HSP70 genes were identified in lettuce (Table 1), which were named LsHSP70-1 to LsHSP70-37. The coding sequences (CDS) ranged from 1948 bp to 6494 bp, protein lengths were between 598 and 701 amino acids, and isoelectric points ranged from 5.07 to 8.48, among which 13 genes with pI > 7, and the rest were acidic (pI < 7). These 37 genes were from seven chromosomes of lettuce, of which 10 LsHSP70 genes were enriched on chromosome 9 (Figure 1). Among these 37 genes LsHSP70-33 had the lowest molecular weight (MW), and LsHSP70-15 had the highest MW. Subcellular localization predictions showed that LsHSP70 genes could be localized in the nucleus, mitochondria, chloroplasts, and endoplasmic reticulum, and 14 of them were predicted to be localized in chloroplasts, which was the organelle that appeared with the highest frequency among the predicted locations.

2.2. Phylogenetic Relationship Analysis of LsHSP70

The phylogenetic tree was constructed by HSP70 from Arabidopsis thaliana, rice, and lettuce (Figure 2). LsHSP70 are categorized into four groups: A, B, C, and D. Group A had the highest number of 21 LsHSP70 genes, while group C contained only LsHSP70-17 and LsHSP70-18, and groups B and D had nine and five LsHSP70 genes each. In addition, there were no AtHSP70 genes in group C, and the OsHSP70 genes were also distant from the LsHSP70 genes, which may indicate that this class of LsHSP70 genes was distantly related to the HSP70 genes of Arabidopsis and rice.

2.3. Gene Structure and Motif Localization Analysis of LsHSP70

Gene structure prediction showed (Figure 3B) that the number of exons in LsHSP70 genes ranged 1–9, and all 19 genes in group A contained two exons except for LsHSP70-1 and LsHSP70-12, which had one and nine exons, respectively. All genes in group B contained two exons except for LsHSP70-14. All genes in group C contained only two exons. The genes in group D contained a high number of exons, containing six or eight exons.
The conserved motifs of LsHSP70 were analyzed by MEME 5.5.7 software, and a total of 15 different motifs were detected (Figure 3C). The vast majority of the genes in group A contained 14 or 15 motifs. All nine LsHSP70s in group B contained 15 motifs. LsHSP70-17 and LsHSP70-18 in group C contained 13 and 14 motifs, respectively. Group D contained the fewest motifs, and LsHSP70-13 contained only seven motifs. All 37 LsHSP70s contained Motif1, Motif2, Motif3, Motif6, Motif8, and Motif11. This indicates that these six motifs may play a central role in the LsHSP70 protein domain. They are not only involved in the basic molecular chaperone function of HSP70, and they may be involved in signal transduction, protein stability and cell stress response.

2.4. Covariance Analysis of LsHSP70

The covariance analysis reveals the evolutionary mechanism of the gene family of the species. Among the 4479 covariate pairs in lettuce (Figure 4), nine pairs of LsHSP70 genes were identified to be in a covariate relationship. This suggests that these nine pairs of genes were formed through large fragment duplication. A higher number of genes with covariance existed on chromosomes 8 and 9 with the LsHSP70-20 covariant with both the LsHSP70-21, LsHSP70-28, and LsHSP70-36, and LsHSP70-36 covariant with LsHSP70-8, LsHSP70-20, and LsHSP70-28.
In addition, the homology of the LsHSP70 gene family was further investigated, the covariances at the gene level in lettuce HSP70, Arabidopsis HSP70, and sunflower HSP70 were analyzed (Figure 5). There were three covariate pairs belonging to LsHSP70 in lettuce and Arabidopsis, which were from chromosome 5 in Arabidopsis and chromosomes 2 and 9 in lettuce, respectively. In comparison with sunflower, there were nine covariate pairs, but again, most of the covariate pairs were on chromosome 9 of lettuce. This suggests that the LsHSP70 gene may be more closely related to sunflower.

2.5. Cis-Acting Element Analysis of LsHSP70

The cis-acting elements of the upstream 2000 bp promoter region of 37 LsHSP70 genes were analyzed (Figure 6). A total of 34 regulatory elements were identified as belonging to four categories. These categories included phytohormones, growth and development, stress response, and light response. The highest number of action elements was related to light response, and the smallest number of elements was related to the growth and development category. Among the phytohormone response elements ABRE, CGTCA and TGACG appeared most frequently as abscisic acid (ABA) and methyl jasmonate (MeJA) response elements, respectively. Among the growth and development-related elements, O2-site and CAT-box appeared with the highest frequency, which were maize protein metabolism and meristematic tissue expression response elements, respectively. The ARE and MBS stress response elements responding to anaerobiosis and drought appeared with higher frequencies. The light response-related elements appeared with similar frequencies, and they were also more evenly distributed among individual LsHSP70 genes. The results suggest that a series of elements with higher frequencies may be closely related to the role of LsHSP70 in different environments.

2.6. Analysis of the Expression Pattern of LsHSP70 Under High-Temperature Stress

The expression levels of the LsHSP70 gene family in response to high-temperature stress were analyzed by transcriptome data (Figure 7). The expression levels of the vast majority of LsHSP70 genes were low at the early stage of stress (2 h of stress). LsHSP70-19, LsHSP70-23, LsHSP70-24, LsHSP70-8, and LsHSP70-14 exhibited faster responses to heat stress. According to the phylogenetic tree, four of the five LsHSP70s belonged to group B, while LsHSP70-19 belonged to group A, and LsHSP70-19 was differentially expressed at its gene level. The expression levels of the majority of the genes were enhanced at the 4th day with the prolongation of stress. The expression levels of seven genes were decreased after the 4th day under high-temperature stress, LsHSP70-36, LsHSP70-9, LsHSP70-13, LsHSP70-10, LsHSP70-4, LsHSP70-6, and LsHSP70-26. However, the expression of LsHSP70-36 was the weakest, and its expression was smaller throughout the stress period. Most of these seven genes belonged to group A. This suggests that LsHSP70 in group A may negatively regulate the tolerance of lettuce under high-temperature stress. Therefore, LsHSP70-19, a more prominent expression pattern in group A, was selected for further study.

2.7. Role of LsHSP70-19 Under High-Temperature Stress

The role of LsHSP70-19 gene on lettuce heat tolerance was further explored. The gene for silencing as well as overexpression was analyzed (Figure 8). We cloned 543 bp and full-length fragments from the CDS region of LsHSP70-19 into pTRV2 and pRI101 vectors, respectively. After Agrobacterium was mediated and validated, the constructs pTRV2-LsHSP70-19 and pRI101-LsHSP70-19 were successfully transferred into lettuce and expressed.
The expression in the silencing lettuce was lower than that in the control (CK), and the expression in the overexpression plants was higher than that in CK. The leaves of silenced lettuce were elongated, and the total fresh weight and shoot fresh weight were significantly lower than CK, while the leaves of overexpression showed no significant change in phenotype with the CK, but the total and shoot fresh weight were significantly higher than CK (Table 2). The above results indicate that the overexpression of LsHSP70-19 can improve the heat resistance of lettuce.

3. Discussion

Lettuce is a typical heat-sensitive vegetable, it is susceptible to high-temperature stress in summer during annual production, and its quality and yield are decreased. Few studies have been found on the HSP70 family in lettuce; therefore, the HSP70 family was characterized using bioinformatics tools and its expression pattern and role were explored under high-temperature stress.
The 37 HSP70 genes were found in lettuce, a significant difference from the number of 18 HSP70 genes in Arabidopsis [15], which may be due to species differences. The 61, 21, and 33 HSP70 genes were found in soybean [18], pumpkin [21], and grape [13], respectively, and their HSP70 genes were not exactly equal in number, either. A total of 113 HSP70 genes were identified in Gossypium hirsutum, G. barbadense, G. arboreum, and G. raimondii. Among them, there were 33 HSP70 genes in G. arboreum and 32 HSP70 genes in G. barbadense [22]. Even for the same family, the number of homologous gene families varies among species due to the size of the genome number and the direction of evolutionary selection [18,23].
The LsHSP70 family in terms of physicochemical properties, phylogenetic relationships, gene structure, and cis-acting element analysis of promoters was analyzed. The LsHSP70 gene family was distributed individually or in clusters on seven chromosomes of lettuce, and LsHSP70 was absent from chromosome 3 and chromosome 7; similarly, none of the chromosomes 3, 9, 13, and 16 of the rowan tree distribute HSP70 family members [24]. No HSP70 gene family members were detected on chromosomes 1 and 12 of Gossypium raimondii [25]. Among the seven chromosomes of cucumber genome, only five chromosomes had 12 CsHSP70 genes, and no CsHSP70 genes were distributed on chromosomes 1 and 6 [26]. The evolutionary process of the plant genome structure is relatively conservative, which may lead to the retention of the HSP70 gene on specific chromosomes, while other chromosomes are not distributed.
Phylogenetically closely related HSP70 genes have the same or similar subcellular localization and gene structure [22]. Most of the genes in group A were localized in mitochondria; all of the genes in group C were localized in the endoplasmic reticulum and nucleus. However, the genes in group D were localized only in mitochondria or chloroplasts. The subcellular localization prediction of Gossypium raimondii showed that it was located in the cytoplasm, endoplasmic reticulum and mitochondria [25]. Comparing the homologous genes of the lettuce HSP70 gene family in rice and Arabidopsis, it can be found that they have similar subcellular localization [15,17].
According to motif analysis, the 21 LsHSP70 genes contained 1–15 motifs, and the vast majority of genes in group A contained 14 or 15 motifs. All LsHSP70 in group B contained 15 motifs. LsHSP70-17 and LsHSP70-18 in group C contained 13 and 14 motifs, respectively. Group D contained 13 motifs. In potato, the number and location of motifs of StHSP70 genes in the same group were more similar [7]. The C-terminal motifs are more conserved than the N-terminal motifs. Unique characteristic motifs exist among different groups. This indicates their evolutionary relationships and different functional divisions [1].
In addition, according to the gene structure analysis, the number of introns in group A does not exceed two except for LsHSP70-12. In addition to LsHSP70-1 and LsHSP70-12 containing one and nine exons, the remaining LsHSP70 genes contain two exons. Group B genes had one or no introns, except that LsHSP70-14 contained only one exon, while the rest contained two exons. Group C contained only two exons. Group D contained six or eight exons. This is consistent with the findings in other species. AtHSP70 genes with a similar number of exons in Arabidopsis thaliana are located in the same organelle [15], and the GmHSP70 gene close to soybean has a similar number of exons [18]. The gene structure will determine the function of the gene to a certain extent. The presence of introns and rapid gene expression are controversial. Because there are few or no introns in plants, it can enhance the immediate activation of gene expression [27]. The genes of groups A and B can respond faster to heat stress compared to groups C and D.
Gene duplication is a common mechanism of gene family expansion, and it increases genetic novelty during plant evolution. It has important implications for genome evolution in plants [28]. Covariance analysis can further demonstrate the replicative relationships of gene families within species and evolutionary similarities between species. Within lettuce, nine gene pairs were collinear, three pairs of genes were collinear with Arabidopsis thaliana, and nine pairs of genes were collinear with sunflower. The sunflower belongs to the same family of Asteraceae crops as lettuce, and the two species have more direct genes between them and are more closely related than lettuce and Arabidopsis. However, LsHSP70-28 was covariant with both Arabidopsis and sunflower, suggesting that this gene’s function is conserved across species. Among the 10 StHSP70 genes in potato, three pairs of tandem genes and two pairs of segmented repeat genes were found [7]. The collinearity analysis of the pumpkin HSP70 gene family showed that there were nine pairs of gene fragment repeats [21]. In the cucumber genome, two CsHSP70 genes formed a fragment repeat [26]. Therefore, species-specific duplication or deletion during evolution may lead to differences in the number of HSP70 family genes between different species.
Cis-acting elements play an important role in gene regulation and expression within promoter regions, and well-regulated promoter cis-acting elements are urgently needed for transgenic plant engineering to achieve controlled expression levels of target genes [29]. An analysis of promoter cis-acting elements of the 37 LsHSP70 genes revealed that phytohormone-responsive and light-responsive elements were the most abundant. A majority of these LsHSP70-responsive elements were associated with ABA, MeJA, and an anaerobic environment. ABRE plays an important role in signaling, ABA activation, and resistance in Arabidopsis [30]. The cis-acting elements indicate that the LsHSP70 gene family plays a key role in responding to different environmental changes such as phytohormones, drought, heat, and light.
The role of LsHSP70 under high-temperature stress was investigated; the expression of this gene family was analyzed under heat stress (2 h of stress) and continuous heat stress (4 days). During heat stress, a small fraction of LsHSP70 was able to rapidly respond to high-temperature changes and enhance their expression with a more than 40-fold increase in the expression of LsHSP70-19. The role of LsHSP70-19 in heat tolerance in lettuce under high-temperature stress was initially verified by VIGS and overexpression After 1 week under high temperature, the fresh weight and root weight of the silenced plants were lower than CK, and the expression of LsHSP70-19 was also lower than CK. After overexpressing LsHSP70-19, the heat tolerance of lettuce was improved, and LsHSP70-19 played a positive regulatory role under high-temperature stress. In Arabidopsis, the expression of AtHSP70 genes in Arabidopsis seedlings was significantly enhanced after 2 h of heat stress [15], and the expression levels of four OsHSP70 genes in rice were also markedly enhanced after 3 h of high heat stress (42 °C) [31]. This suggests the existence of a number of HSP70 genes that can rapidly respond to high-temperature stress and enhance their expression levels. However, in persistent heat stress, the expression levels of most of the genes increased with the prolongation of stress time, and only a few of them remained unchanged or decreased after heat treatment. This suggests that there may be a functional division of labor among LsHSP70 genes in response to heat stress.

4. Materials and Methods

4.1. Plant Materials and Treatments

The plant material used was heat-sensitive lettuce cultivar ‘Beisansheng No. 3’ by the Beijing University of Agriculture. The cultivate conditions were incubated at 20 ± 2 °C (day)/13 ± 2 °C (night), with a photoperiod of 14 h (day)/10 h (night), relative humidity of 60 ± 5%, and light intensity of 12,000 Lux. Lettuce was treated in 5 leaves. The treatment conditions were room temperature (CK) 20 ± 2 °C (day)/13 ± 2 °C (night), high temperature (H) 33 ± 2 °C (day)/25 ± 2 °C (night), and the rest of the conditions remained the same. Samples of lettuce leaves were taken at 2 h and 4 days of high-temperature stress with each treatment replicated at least 6 times.

4.2. Identification and Physicochemical Properties of HSP70 Family Members in Lettuce

The whole genome sequence of lettuce was downloaded from the Ensemble Plants database (https://plants.ensembl.org/index.html; accessed on 11 July 2022) as a basis to establish a local protein database. Overall, 18 HSP70 genes of Arabidopsis thaliana were obtained, and the constructed lettuce database was searched with BLASTP by the structural sequence of Arabidopsis thaliana as a query benchmark. The genes with an E-value of <0.05 were selected after deleting duplicates. The preliminarily screened lettuce HSP70 genes were subsequently confirmed by HMM and the Pfam database to confirm the presence of the HSP70 structural domain in each of them. Eventually, a total of 37 HSP70 genes were confirmed as LsHSP70 genes in lettuce.
The ProtParam tool (https://web.expasy.org/protparam; accessed on 30 November 2022) was used to predict the physical and chemical properties of lettuce HSP70, including the number of amino acids (aa), the isoelectric point (pI), and the molecular weight (MW) of LsHSP70, and the results are demonstrated in Table 1.
Cell-PLoc 2.0 was used to predict subcellular localization (http://www.csbio.sjtu.edu.cn/bioinf/Cell-PLoc-2; accessed on 5 December 2022). HSP70 members for the remaining species were obtained from the corresponding plant databases, and data for each species were downloaded from Phytozome V13 (https://phytozome-next.jgi.doe.gov; accessed on 11 July 2022).

4.3. Chromosomal Localization of LsHSP70 and Construction of Phylogenetic Tree

By querying the annotation file of lettuce genome, the chromosome location and direction of HSP70 were obtained, and the gene density in chromosome was obtained by TBtools windows-x64_1_113. The chromosome location was visualized, and the chromosome location of 37 LsHSP70 genes was completed. The construction of the phylogenetic tree was performed by MEGA 11.0 software. ClustalW 2.1 was utilized for multiple sequence comparison of lettuce, Arabidopsis thaliana, and rice. The phylogenetic tree was constructed by the neighbor-joining (NJ) method based on 1000 bootstrap replicates, and the output phylogenetic tree was finally colored and beautified using the iTOL website (https://itol.embl.de; accessed on 15 October 2021).

4.4. Gene Structure and Motif Analysis of LsHSP70

The 37 CDS of LsHSP70 were mapped to the gene structure of lettuce HSP70 by TBtools windows-x64_1_113 software. The conserved motifs of LsHSP70 were identified using a MEME tool (https://meme-suite.org/meme/info/status?service=MEME&id=appMEME_5.5.11676975855714812268290; accessed on 18 October 2021). A total of 15 motifs were selected, the characteristics of each motif were retained, and finally the motifs of 37 LsHSP70 genes were mapped by TBtools windows-x64_1_113.

4.5. Analysis of Covariance

The established database and TBtools windows-x64_1_113 software were used to obtain the required files such as the chromosome skeleton, gene density, and the location of LsHSP70 genes in lettuce. Circos plots were used to visualize the homology between LsHSP70 genes. The collinearity analysis of genes among lettuce, Arabidopsis thaliana and sunflower was performed to analyze the homologous relationship between their HSP70s. The whole genome and annotation information of sunflower was from the phytozome database (https://phytozome-next.jgi.doe.gov; accessed on 7 February 2023).

4.6. Analysis of Promoter Action Elements of LsHSP70 Gene

A 2000 bp region upstream of LsHSP70 was selected in the established lettuce database, and the cis-acting elements were identified and analyzed in this region. Data on cis-acting elements were from the PlantCARE online database (https://bioinformatics.psb.ugent.be/webtools/plantcare/html/; accessed on 20 October 2021). The identified elements were visualized by TBtools windows-x64_1_113.

4.7. Expression Analysis of LsHSP70-19 Under High-Temperature Stress

Fresh lettuce leaves were selected as materials for RNA extraction. The sample was filled in a 2 mL reagent tube containing 3 steel balls with a diameter of 2 mm. The tubes were labeled with the sample names and stored at −80 °C. Total RNA was extracted according to the Quick RNA Isolation Kit (Quick RNA Isolation Kit, Huayueyang, Beijing, China). The first cDNA strand was synthesized by a reverse transcription kit (Tiangen, Beijing, China). The resulting cDNA was used as a template for quantitative real-time polymerase reaction (qRT-PCR) with 18S as the internal reference gene. qRT-PCR was performed by TB Green Premix Ex TaqTM II (TaKaRa, Beijing, China) on a CFX real-time PCR detection system instrument (Bio-Rad Laboratories, Hercules, CA, USA). The internal reference gene was 18S, and the reaction system was 20 μL, including 9 μL TB Green Premix Ex TaqTM II (TaKaRa), 7 μL ddH2O, 1 μL forward primer, 1 μL reverse primer and 2 μL cDNA template. Expression was calculated by the 2−ΔΔCt method, and three technical replicates were performed for each sample. The expression levels of LsHSP70 were extracted from previous transcriptome data.

4.8. Construction of VIGS Gene Silencing and Overexpression Vector

The 543bp sequence of the CDS region of LsHSP70-19 was amplified by a VIGS-LsHSP70-19-F/R primer and pRI101-LsHSP70-19-F/R primer (Table S1). The pTRV2 vector was linearized by KpnI, and the pRI101 vector was linearized by BamHI and NdeI. A homologous recombination of amplified bands and linearized vectors was performed using a ClonExpressII One Step Cloning Kit (Vazyme Biotech, Beijing, China). The recombinant plasmids were transferred into DH5α competent cells, sequenced correctly, and then transferred into GV3101 chemically competitive cells (Weidi, Biotechnology, Shanghai, China). Before VIGS injection, pTRV1 and pTRV2 were mixed in equal volumes. Each treatment had 30 lettuce plants. Four treatment groups were established: blank control (no injection), empty vector (injection with equal pTRV2 and pTRV1), silencing (injection with equal pTRV2-LsHSP70-19 and pTRV1), and overexpression (injection of pRI101-LsHSP70-19). Seven days after injection, high-temperature treatment was performed. After 14 days of treatment, three plants with consistent growth were selected from each treatment group, and new leaves were taken. DNA was extracted from each plant in triplicate to verify the positive transformation. Samples were collected from different parts of each plant in triplicate. RNA was extracted to assess vector expression, determine the expression level of LsHSP70-19, and measure the growth indexes of each group.

5. Conclusions

The 37 LsHSP70 gene family members were identified from the whole genome of lettuce by the bioinformatics method, and their physicochemical properties, chromosomal localization, phylogenetic relationship, gene structure, and cis-acting element analysis of the promoter were analyzed. To understand the response of LsHSP70 to high-temperature stress, we analyzed its expression patterns by transcriptome data. LsHSP70-19, with a more prominent expression pattern, was selected to preliminarily verify its positive regulatory role under high-temperature stress through virus-induced gene silencing and overexpression. The molecular function of LsHSP70 under high-temperature stress requires further investigation.

Supplementary Materials

The following supporting information can be downloaded at https://www.mdpi.com/article/10.3390/ijms26010102/s1.

Author Contributions

Data curation, Q.W., Y.X. and W.S.; investigation, writing—original draft, Q.W. and W.S.; formal analysis, Q.W., W.S. and Y.D.; conceptualization, C.L.; writing—review and editing, C.L., H.W. and J.H.; funding acquisition, C.L. and Y.H. All authors have read and agreed to the published version of the manuscript.

Funding

This work was supported by the Beijing Agricultural Science and Technology Project for Rural Revitalization (20250107).

Institutional Review Board Statement

Not applicable.

Informed Consent Statement

Not applicable.

Data Availability Statement

All of the data are contained within the article.

Conflicts of Interest

The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.

References

  1. Wang, T.Z.; Liu, M.J.; Wu, Y.; Tian, Y.F.; Han, Y.Y.; Liu, C.J.; Hao, J.H.; Fan, S.X. Genome-Wide Identification and Expression Analysis of MAPK Gene Family in Lettuce (Lactuca sativa L.) and Functional Analysis of LsMAPK4 in High—Temperature-Induced Bolting. Int. J. Mol. Sci. 2022, 23, 11129. [Google Scholar] [CrossRef] [PubMed]
  2. Guo, M.; Liu, J.H.; Lu, J.P.; Zhai, Y.F.; Wang, H.; Gong, Z.H.; Wang, S.B.; Lu, M.H. Genome-wide analysis of the CaHsp20 gene family in pepper: Comprehensive sequence and expression profile analysis under heat stress. Front. Plant Sci. 2015, 6, 806. [Google Scholar] [CrossRef]
  3. Johnson, J.L.; Brown, C. Plasticity of the Hsp90 chaperone machine in divergent eukaryotic organisms. Cell Stress Chaperones 2009, 14, 83–94. [Google Scholar] [CrossRef]
  4. Wu, J.P. Cloning and functional identification of chrysanthemum heat shock protein gene CmHSP90.5. Master’s Thesis, Nanjing Agricultural University, Nanjing, China, 2020. [Google Scholar]
  5. Song, Z.; Pan, F.; Lou, X.; Wang, D.; Yang, C.; Zhang, B.; Zhang, H. Genome-wide identification and characterization of Hsp70 gene family in Nicotiana tabacum. Mol. Biol. Rep. 2019, 46, 1941–1954. [Google Scholar] [CrossRef] [PubMed]
  6. Flaherty, K.M.; DeLuca-Flaherty, C.; McKay, D.B. Three-dimensional structure of the ATPase fragment of a 70K heat-shock cognate protein. Nature 1990, 346, 623–628. [Google Scholar] [CrossRef]
  7. Liu, J.; Pang, X.; Cheng, Y.; Yin, Y.; Zhang, Q.; Su, W.; Hu, B.; Guo, Q.; Ha, S.; Zhang, J.; et al. The Hsp70 Gene Family in Solanum tuberosum: Genome-Wide Identification, Phylogeny, and Expression Patterns. Sci. Rep. 2018, 8, 16628. [Google Scholar] [CrossRef]
  8. Guo, M.; Liu, J.H.; Ma, X.; Zhai, Y.F.; Gong, Z.H.; Lu, M.H. Genome-wide analysis of the Hsp70 family genes in pepper (Capsicum annuum L.) and functional identification of CaHsp70-2 involvement in heat stress. Plant Sci. 2016, 252, 246–256. [Google Scholar] [CrossRef] [PubMed]
  9. Sung, D.Y.; Guy, C.L. Physiological and molecular assessment of altered expression of Hsc70-1 in Arabidopsis. Evidence for pleiotropic consequences. Plant Physiol. 2003, 132, 979–987. [Google Scholar] [CrossRef] [PubMed]
  10. Cho, E.K.; Hong, C.B. Over-expression of tobacco NtHSP70-1 contributes to drought-stress tolerance in plants. Plant Cell Rep. 2006, 25, 349–358. [Google Scholar] [CrossRef] [PubMed]
  11. Duan, Y.H.; Guo, J.; Ding, K.; Wang, S.J.; Zhang, H.; Dai, X.W.; Chen, Y.Y.; Govers, F.; Huang, L.L.; Kang, Z.S. Characterization of a wheat HSP70 gene and its expression in response to stripe rust infection and abiotic stresses. Mol. Biol. Rep. 2011, 38, 301–307. [Google Scholar] [CrossRef]
  12. Kim, S.R.; An, G. Rice chloroplast-localized heat shock protein 70, OsHsp70CP1, is essential for chloroplast development under high-temperature conditions. J. Plant Physiol. 2013, 170, 854–863. [Google Scholar] [CrossRef]
  13. Xinna, L.; Haiyang, C.; Shenchang, L.; Lijun, W. Genome-Wide Identification of the Hsp70 Gene Family in Grape and Their Expression Profile during Abiotic Stress. Horticulturae 2022, 8, 743. [Google Scholar] [CrossRef]
  14. Mengru, Y.; Rongqian, H.; Aiping, S.; Zhiyong, G.; Fadi, C.; Jiafu, J. Genome-Wide Identification and Expression Analysis of HSP70 Gene Family in Chrysanthemum lavandulifolium under Heat Stress. Horticulturae 2023, 9, 238. [Google Scholar] [CrossRef]
  15. Lin, B.L.; Wang, J.S.; Liu, H.C.; Chen, R.W.; Meyer, Y.; Barakat, A.; Delseny, M. Genomic analysis of the Hsp70 superfamily in Arabidopsis thaliana. Cell Stress Chaperones 2001, 6, 201–208. [Google Scholar] [CrossRef]
  16. Guy, C.L.; Li, Q.B. The organization and evolution of the spinach stress 70 molecular chaperone gene family. Plant Cell 1998, 10, 539–556. [Google Scholar] [CrossRef] [PubMed]
  17. Jung, K.H.; Gho, H.J.; Nguyen, M.X.; Kim, S.R.; An, G. Genome-wide expression analysis of HSP70 family genes in rice and identification of a cytosolic HSP70 gene highly induced under heat stress. Funct. Integr. Genomics 2013, 13, 391–402. [Google Scholar] [CrossRef]
  18. Zhang, L.; Zhao, H.K.; Dong, Q.L.; Zhang, Y.Y.; Wang, Y.M.; Li, H.Y.; Xing, G.J.; Li, Q.Y.; Dong, Y.S. Genome-wide analysis and expression profiling under heat and drought treatments of HSP70 gene family in soybean (Glycine max L.). Front. Plant Sci. 2015, 6, 773. [Google Scholar] [CrossRef]
  19. Ni, Z.Y.; Liu, N.; Yu, Y.H.; Bi, C.X.; Chen, Q.J.; Qu, Y.Y. The cotton 70-kDa heat shock protein GhHSP70-26 plays a positive role in the drought stress response. Environ. Exp. Bot. 2021, 191, 104628. [Google Scholar] [CrossRef]
  20. Yang, X.; Wei, S.; Liu, B.; Guo, D.; Zheng, B.; Feng, L.; Liu, Y.; Tomás-Barberán, F.A.; Luo, L.; Huang, D. A novel integrated non-targeted metabolomic analysis reveals significant metabolite variations between different lettuce (Lactuca sativa L.) varieties. Hortic. Res. 2018, 5, 33. [Google Scholar] [CrossRef] [PubMed]
  21. Davoudi, M.; Chen, J.; Lou, Q. Genome-Wide Identification and Expression Analysis of Heat Shock Protein 70 (HSP70) Gene Family in Pumpkin (Cucurbita moschata L.) Rootstock under Drought Stress Suggested the Potential Role of these Chaperones in Stress Tolerance. Int. J. Mol. Sci. 2022, 23, 1918. [Google Scholar] [CrossRef] [PubMed]
  22. Rehman, A.; Atif, R.M.; Qayyum, A.; Du, X.; Hinze, L.; Azhar, M.T. Genome-wide identification and characterization of HSP70 gene family in four species of cotton. Genomics 2020, 112, 4442–4453. [Google Scholar] [CrossRef]
  23. Wang, X.R.; Wang, C.; Ban, F.X.; Zhu, D.T.; Liu, S.S.; Wang, X.W. Genome-wide identification and characterization of HSP gene superfamily in whitefly (Bemisia tabaci) and expression profiling analysis under temperature stress. Insect Sci. 2019, 26, 44–57. [Google Scholar] [CrossRef] [PubMed]
  24. Li, Y.Y.; Zhang, Z.R.; Di, Z.X.; Qi, X.Y.; Zhang, Y.; Wang, C.; Lu, Y.Z.; Zheng, J. Identification of HSP70 gene family in Sorbus pohuashanensis and its expression analysis in response to abiotic stress. Genom. Appl. Biol. 2022, 41, 1973–1984. [Google Scholar] [CrossRef]
  25. Zhang, Y.T. Evolution of HSP70 gene family and functional analysis of GhHSP70-20 in Gossypium raimondii. Master’s Thesis, Zhejiang Agricultural and Forestry University, Hangzhou, China, 2014. [Google Scholar]
  26. 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. [Google Scholar] [CrossRef]
  27. Parra, G.; Bradnam, K.; Rose, A.B.; Korf, I. Comparative and functional analysis of intron-mediated enhancement signals reveals conserved features among plants. Nucleic Acids Res. 2011, 39, 5328–5337. [Google Scholar] [CrossRef] [PubMed]
  28. Magadum, S.; Banerjee, U.; Murugan, P.; Gangapur, D.; Ravikesavan, R. Gene duplication as a major force in evolution. J. Genet. 2013, 92, 155–161. [Google Scholar] [CrossRef] [PubMed]
  29. Mishra, A.; Tanna, B. Halophytes: Potential Resources for Salt Stress Tolerance Genes and Promoters. Front. Plant Sci. 2017, 8, 829. [Google Scholar] [CrossRef]
  30. Yoshida, T.; Fujita, Y.; Sayama, H.; Kidokoro, S.; Maruyama, K.; Mizoi, J.; Shinozaki, K.; Yamaguchi-Shinozaki, K. AREB1, AREB2, and ABF3 are master transcription factors that cooperatively regulate ABRE-dependent ABA signaling involved in drought stress tolerance and require ABA for full activation. Plant J. 2010, 61, 672–685. [Google Scholar] [CrossRef] [PubMed]
  31. Ye, S.F.; Yu, S.W.; Shu, L.B.; Wu, J.H.; Wu, A.Z.; Luo, L.J. Expression profile analysis of 9 heat shock protein genes throughout the life cycle and under abiotic stress in rice. Chin. Sci. Bull. 2012, 57, 336–347. [Google Scholar] [CrossRef]
Ijms 26 00102 g001
Figure 1. Chromosome localization of LsHSP70. The top yellow text represents the number of chromosomes, the red text represents the gene name, and the scale on the left represents the chromosome size.
Figure 1. Chromosome localization of LsHSP70. The top yellow text represents the number of chromosomes, the red text represents the gene name, and the scale on the left represents the chromosome size.
Ijms 26 00102 g001
Ijms 26 00102 g002
Figure 2. Phylogenetic tree of HSP70 gene family in lettuce, Arabidopsis thaliana and rice. Lettuce genes are shown in black, Arabidopsis genes are shown in red, and rice genes are shown in yellow.
Figure 2. Phylogenetic tree of HSP70 gene family in lettuce, Arabidopsis thaliana and rice. Lettuce genes are shown in black, Arabidopsis genes are shown in red, and rice genes are shown in yellow.
Ijms 26 00102 g002
Ijms 26 00102 g003
Figure 3. Gene structure of LsHSP70 and motif analysis. (A) LsHSP70 evolutionary tree; (B) gene structure; (C) motif distribution; (D) motif pattern. Figure 3D represents the sequence identity of the motif. Different colors correspond to different motifs, and different letter sizes represent different frequency matrices.
Figure 3. Gene structure of LsHSP70 and motif analysis. (A) LsHSP70 evolutionary tree; (B) gene structure; (C) motif distribution; (D) motif pattern. Figure 3D represents the sequence identity of the motif. Different colors correspond to different motifs, and different letter sizes represent different frequency matrices.
Ijms 26 00102 g003
Ijms 26 00102 g004
Figure 4. Collinearity analysis of LsHSP70 gene family. The gray lines in the background represent homogenous pairs in the entire lettuce genome, and the black lines represent homogenous pairs in the LsHSP70. The information represented by each circle in the figure from the inside out was the genome density heat map, genome density linear map, chromosome name, and LsHSP70 chromosome location labeling.
Figure 4. Collinearity analysis of LsHSP70 gene family. The gray lines in the background represent homogenous pairs in the entire lettuce genome, and the black lines represent homogenous pairs in the LsHSP70. The information represented by each circle in the figure from the inside out was the genome density heat map, genome density linear map, chromosome name, and LsHSP70 chromosome location labeling.
Ijms 26 00102 g004
Ijms 26 00102 g005
Figure 5. Collinearity analysis of lettuce with Arabidopsis and sunflower. The gray line in the background represents all collinear pairs of lettuce with Arabidopsis and sunflower at the genome level. The black line represents a common pair of HSP70 family members belonging to the three species.
Figure 5. Collinearity analysis of lettuce with Arabidopsis and sunflower. The gray line in the background represents all collinear pairs of lettuce with Arabidopsis and sunflower at the genome level. The black line represents a common pair of HSP70 family members belonging to the three species.
Ijms 26 00102 g005
Ijms 26 00102 g006aIjms 26 00102 g006b
Figure 6. LsHSP70 promoter analysis. Location map of cis-acting elements related to plant hormones (A), plant development (B), stress response (C), and light response (D). (E) Heat map of the number of instantaneous cis-acting components.
Figure 6. LsHSP70 promoter analysis. Location map of cis-acting elements related to plant hormones (A), plant development (B), stress response (C), and light response (D). (E) Heat map of the number of instantaneous cis-acting components.
Ijms 26 00102 g006aIjms 26 00102 g006b
Ijms 26 00102 g007
Figure 7. Expression of LsHSP70 at high temperature. CK, room temperature treatment; H, high-temperature treatment; 2H, treatment two hours; 4D, treatment four days.
Figure 7. Expression of LsHSP70 at high temperature. CK, room temperature treatment; H, high-temperature treatment; 2H, treatment two hours; 4D, treatment four days.
Ijms 26 00102 g007
Ijms 26 00102 g008
Figure 8. Role of LsHSP70-19 at high temperature. (A) Cloning of LsHSP70-19; 1–4 in the figure represent 4 replicates. (B) Phenotypic map of lettuce under different treatments. (C) The expression of LsHSP70-19 under different treatments. CK, control plants; Silence, silent plants; OE, overexpression plants. Different letters represent significant differences, determined by one-way ANOVA and Duncan’s test. p < 0.05.
Figure 8. Role of LsHSP70-19 at high temperature. (A) Cloning of LsHSP70-19; 1–4 in the figure represent 4 replicates. (B) Phenotypic map of lettuce under different treatments. (C) The expression of LsHSP70-19 under different treatments. CK, control plants; Silence, silent plants; OE, overexpression plants. Different letters represent significant differences, determined by one-way ANOVA and Duncan’s test. p < 0.05.
Ijms 26 00102 g008
Table 1. Physical and chemical properties of LsHSP70.
Table 1. Physical and chemical properties of LsHSP70.
Gene NameLocus IDChromosomal LocationLength (aa)MW (kDa)pISubcellular Localization
ChrChr-StartChr-EndDirection
LsHSP70-1Lsat_1_v5_gn_1_873401109,889,259109,895,753-645725.18Endoplasmic reticulum
LsHSP70-2Lsat_1_v5_gn_1_917001120,103,665120,109,556+617698.11Endoplasmic reticulum
LsHSP70-4Lsat_1_v5_gn_1_933211121,003,737121,009,958+617687.84Endoplasmic reticulum
LsHSP70-3Lsat_1_v5_gn_1_911411120,849,862120,855,133+617696.51Mitochondrion
LsHSP70-5Lsat_1_v5_gn_1_912411121,066,030121,067,978-617697.53Endoplasmic reticulum
LsHSP70-6Lsat_1_v5_gn_1_913811121,849,518121,855,607+617698.48Endoplasmic reticulum
LsHSP70-8Lsat_1_v5_gn_2_1166402195,818,062195,820,849+648715.07Nucleus
LsHSP70-7Lsat_1_v5_gn_2_1057202183,329,797183,331,788+626705.30Chloroplast
LsHSP70-11Lsat_1_v5_gn_4_726004114,729,310114,731,461+614698.01Mitochondrion
LsHSP70-9Lsat_1_v5_gn_4_50420474,953,55074,956,176+614698.32Chloroplast
LsHSP70-13Lsat_1_v5_gn_4_1731804350,529,997350,533,516+632716.21Mitochondrion
LsHSP70-12Lsat_1_v5_gn_4_1373014264,836,010264,839,010-636708.28Endoplasmic reticulum Mitochondrion
LsHSP70-10Lsat_1_v5_gn_4_725804114,717,746114,719,920+616698.20Mitochondrion
LsHSP70-15Lsat_1_v5_gn_5_1046015223,025,473223,028,553-701755.14Chloroplast
LsHSP70-16Lsat_1_v5_gn_5_1109015230,527,527230,530,603-680735.78Mitochondrion
LsHSP70-14Lsat_1_v5_gn_5_894405198,889,223198,891,499+649715.18Nucleus
LsHSP70-18Lsat_1_v5_gn_6_835206137,995,823137,998,823-661735.08Endoplasmic reticulum Nucleus
LsHSP70-19Lsat_1_v5_gn_6_1126216184,461,293184,464,461-690745.11Chloroplast
LsHSP70-17Lsat_1_v5_gn_6_835816137,948,656137,951,646+623695.84Endoplasmic reticulum Nucleus
LsHSP70-23Lsat_1_v5_gn_8_780218111,243,941111,246,426+649715.13Nucleus
LsHSP70-26Lsat_1_v5_gn_8_1035218156,999,462157,001,748-610686.47Chloroplast
LsHSP70-20Lsat_1_v5_gn_8_15681820,742,29820,744,707+651725.58Nucleus
LsHSP70-21Lsat_1_v5_gn_8_781418111,049,782111,052,054+646715.12Chloroplast
LsHSP70-27Lsat_1_v5_gn_8_1039018157,310,376157,312,452-613688.35Mitochondrion
LsHSP70-24Lsat_1_v5_gn_8_780008111,278,374111,281,033-648715.15Endoplasmic reticulum Mitochondrion
LsHSP70-25Lsat_1_v5_gn_8_1044418156,970,950156,973,039+614688.47Mitochondrion
LsHSP70-22Lsat_1_v5_gn_8_781008111,121,876111,124,251-646715.12Chloroplast
LsHSP70-33Lsat_1_v5_gn_9_36681940,914,39240,916,855-598668.24Chloroplast
LsHSP70-28Lsat_1_v5_gn_9_22920925,664,74225,667,641+647715.11Chloroplast
LsHSP70-36Lsat_1_v5_gn_9_65381983,179,84583,182,256+647715.2Chloroplast
LsHSP70-35Lsat_1_v5_gn_9_36981941,045,21541,047,623-610688.02Chloroplast
LsHSP70-31Lsat_1_v5_gn_9_36560940,874,94240,877,359+635718.55Endoplasmic reticulum Mitochondrion
LsHSP70-29Lsat_1_v5_gn_9_34760936,912,22436,914,256+613685.08Mitochondrion Nucleus
LsHSP70-34Lsat_1_v5_gn_9_36861940,973,00640,975,340-610685.56Chloroplast
LsHSP70-32Lsat_1_v5_gn_9_36600940,892,96140,895,271+608675.73Chloroplast Nucleus
LsHSP70-37Lsat_1_v5_gn_9_1136819185,253,863185,258,114-682735.89Mitochondrion
LsHSP70-30Lsat_1_v5_gn_9_36480940,736,15040,738,239+610686.20Chloroplast Nucleus
Table 2. Effects of LsHSP70-19 transient expression or silencing on growth indices of lettuce.
Table 2. Effects of LsHSP70-19 transient expression or silencing on growth indices of lettuce.
TreatmentFresh Weight (g)Plant Height (cm)Root Length (cm)Shoot Weight (g)Root Weight (g)
CK14.49 ± 1.05 b17.97 ± 2.40 a21.6 ± 1.67 b9.89 ± 0.84 b2.34 ± 0.50 a
Overexpression19.5 ± 1.22 a21.67 ± 2.00 a27.53 ± 2.21 a17.31 ± 0.67 a2.32 ± 0.92 a
Silence10.44 ± 0.91 c19.63 ± 0.78 a27.67 ± 3.21 a7.26 ± 0.12 c1.66 ± 0.36 a
Note: Different letters represent significant differences determined by one-way ANOVA and Duncan’s test. p < 0.05.
Disclaimer/Publisher’s Note: The statements, opinions and data contained in all publications are solely those of the individual author(s) and contributor(s) and not of MDPI and/or the editor(s). MDPI and/or the editor(s) disclaim responsibility for any injury to people or property resulting from any ideas, methods, instructions or products referred to in the content.

Share and Cite

MDPI and ACS Style

Wang, Q.; Sun, W.; Duan, Y.; Xu, Y.; Wang, H.; Hao, J.; Han, Y.; Liu, C. Genome-Wide Identification and Expression Analysis of HSP70 Gene Family Under High-Temperature Stress in Lettuce (Lactuca sativa L.). Int. J. Mol. Sci. 2025, 26, 102. https://doi.org/10.3390/ijms26010102

AMA Style

Wang Q, Sun W, Duan Y, Xu Y, Wang H, Hao J, Han Y, Liu C. Genome-Wide Identification and Expression Analysis of HSP70 Gene Family Under High-Temperature Stress in Lettuce (Lactuca sativa L.). International Journal of Molecular Sciences. 2025; 26(1):102. https://doi.org/10.3390/ijms26010102

Chicago/Turabian Style

Wang, Qian, Wenjing Sun, Yipei Duan, Yikun Xu, Huiyu Wang, Jinghong Hao, Yingyan Han, and Chaojie Liu. 2025. "Genome-Wide Identification and Expression Analysis of HSP70 Gene Family Under High-Temperature Stress in Lettuce (Lactuca sativa L.)" International Journal of Molecular Sciences 26, no. 1: 102. https://doi.org/10.3390/ijms26010102

APA Style

Wang, Q., Sun, W., Duan, Y., Xu, Y., Wang, H., Hao, J., Han, Y., & Liu, C. (2025). Genome-Wide Identification and Expression Analysis of HSP70 Gene Family Under High-Temperature Stress in Lettuce (Lactuca sativa L.). International Journal of Molecular Sciences, 26(1), 102. https://doi.org/10.3390/ijms26010102

Note that from the first issue of 2016, this journal uses article numbers instead of page numbers. See further details here.

Article Metrics

Back to TopTop