1. Introduction
The two-spotted spider mite, Tetranychus urticae (Koch), represents a pervasive global threat to agricultural productivity [1], wreaking havoc on crops, fruit trees, and vegetables [2,3,4], particularly in greenhouse and controlled environment agriculture settings [5]. These minuscule pests take up residence on the undersides of plant leaves, spinning intricate webs to create microhabitats that shield them from environmental stressors, ward off predators, enable pheromonal communication, and facilitate dispersion [6]. During the scorching summer months, natural ecosystems and controlled agricultural environments contend with soaring temperatures, which provide ideal conditions for the proliferation of T. urticae populations [7]. Elevated temperatures not only result in more giant adult female mites and increased egg production but also diminish the size of individual eggs [8]. The remarkable adaptability of T. urticae to such high-temperature stressors inflicts significant damage to crops, leading to substantial economic losses and jeopardizing food security.
In an effort to unravel the adaptive strategies employed by T. urticae to cope with high-temperature stress, our previous research uncovered compelling insights. We observed a marked rise in total antioxidant capacity (T-AOC) and the activity levels of three key antioxidant enzymes following short-term heat exposure in T. urticae. This phenomenon suggests that high temperatures induce oxidative stress in these mites, triggering an accumulation of reactive oxygen species (ROS) [9]. However, the heightened activity of antioxidant enzymes effectively scavenges these harmful ROS, bolstering T. urticae’s resilience to high temperatures.
Too high or too low temperature, too strong or weak light intensity, and ultraviolet radiation will cause oxidative stress in organisms [10]. When oxidative stress occurs in the body, many reactive oxygen species (ROS) (O2−, OH−, and H2O2) will be generated immediately [11]. ROS will increase membrane permeability, lipid peroxidation, and cell apoptosis, harming organisms [12]. Antioxidant enzymes play an essential role in coping with oxidative stress and regulating apoptosis and longevity. Superoxide dismutases (SOD), peroxidase (Prx), catalase (CAT), Glutathione peroxidase (GPX), and Glutathione S-transferase (GST) have been reported as the key antioxidant enzymes involved in coping with high-temperature stress in mites and insects [13]. ROS first reacts with SOD to produce H2O2 and O2 [14], and then H2O2 can combine with Prx and CAT to produce H2O and O2 on the one hand [15]. H2O2 can also combine with Glutathione (GSH) and GST to produce water and other nontoxic substances under the catalysis of GPX, on the other hand, to complete the decomposition of H2O2 [16].
GSTs are vital multifunctional proteins within organisms, wielding the power to engage in detoxification and substance metabolism [17]. They protect against exogenous pesticides [18], plant secondary substances [19], and ROS damage [20]. Typically existing as dimers, GSTs catalyze the conjugation of reduced glutathione to the electrophilic groups of endogenous or exogenous substances [21]. This catalytic process aids in the expulsion of harmful substances from the organism, fortifying it against poisoning [22]. Beyond their detoxifying prowess, GSTs also play a pivotal role in neutralizing highly oxidizing ROS such as O2−, OH−, and H2O2, as well as lipid peroxides like malondialdehyde (MDA) [23], thus safeguarding organisms from ROS-induced damage [24]. Although the metabolic mechanism of chemical pesticides garners significant attention in GST studies, scant research delves into the role of GSTs in conferring bioheat resistance.
The publication of the whole genome of T. urticae in 2011 unveiled the presence of 31 GST genes in its genetic blueprint. These include 16 Delta family GSTs, 12 Mu family GSTs, 2 Omega family GSTs, and 1 Theta family GST [25]. To unearth whether GST genes participate in T. urticae’s heat resistance mechanism, we subjected adult female T. urticae to transcriptomic sequencing following short-term high-temperature stress (with 25 °C as the negative control). Our study seamlessly integrated whole-genome and transcriptomic data, identifying six GST genes exhibiting up-regulated expression levels post short-term heat shock. The bioinformatic analysis of these genes unveiled their distinctive characteristics. Subsequently, real-time quantitative PCR (RT-qPCR) was employed to scrutinize the expression patterns of these genes in response to short-term high-temperature stress. This endeavor furnishes a foundational framework for dissecting the mechanisms underpinning T. urticae’s heat resistance and devising effective preventive measures against it.
2. Materials and Methods
2.1. Mite Colony
The Tetranychus urticae population utilized in this research originated from the Laboratory of Insect Systematic and Biodiversity at Gansu Agricultural University in Lanzhou, Gansu, China. Since 2012, these mites have been maintained without pesticide exposure or temperature extremes. They were cultivated on fresh bean plants (Phaseolus vulgaris L.) within controlled climate chambers set at 25 ± 1 °C, with a relative humidity of 60 ± 5%, and subjected to an L16: D8 photoperiod, all under pesticide-free conditions.
2.2. Selection of GST Genes
From the comprehensive data set encompassing 31 GST genes as reported in the whole genome information and the outcomes of transcriptome sequencing (Accession number: PRJNA1073827), we identified GST genes exhibiting up-regulated expression levels through the analysis of differential expressed genes (DEGs). The Log2FC and p-value were calculated using the DEseq2 method, and data visualization was performed using GraphPad Prism (version 8.0).
2.3. Cloning the CDSs of GST Genes
The coding sequences (CDSs) of GST genes were determined utilizing Open Reading Frame Finder (ORF) (
2.4. Bioinformatic Analysis and Identification of GST Genes
The molecular weights and theoretical isoelectric points were calculated using the ExPASy ProtParam tool (
2.5. Structural Characterization of the Coding Region of 6 GST Genes
The structural analysis of GST genes was forecasted using NCBI’s online Conserved Domain Search Service (CD Search) (
2.6. Expression of GST Transcripts
In this study, we investigated the transcriptional expression of antioxidant enzyme genes via RT-qPCR. Furthermore, the α-tubulin gene (Accession number: JN881327.1) for normalization, and the primer sequences utilized are detailed in Table S2. Three hundred adult females were carefully collected with a small brush, transferred into 1.5 mL microtubes, and placed in an artificial control chamber. Samples underwent treatment at varying temperatures (36, 39, and 42 °C) for different durations (2, 4, and 6 h), all within controlled humidity conditions (RH 80 ± 5%). Following treatment, the samples were promptly frozen with liquid nitrogen and preserved at −80 °C in a refrigerator. Adult females reared at 25 °C for each treatment served as negative controls. RNA extraction from each sample was performed using Trizol reagent (Thermo Fisher Scientific, New York, NY, USA), and the quantity and quality of RNA samples were evaluated using a Thermo Scientific NanoDropTM 2000 UV-VIS Spectrophotometer (Thermo Fisher Scientific, New York, NY, USA). Each treatment involved the extraction of RNA from three hundred mites, with five replicates for each treatment. Approximately 1 μg of RNA from each sample was reverse-transcribed into cDNA using the PrimerScriptTM RT reagent Kit with gDNA Eraser (TaKaRa, Dalian, China). RT-qPCR analysis was performed using the ABI QuantStudio 5 Real-Time PCR system (Thermo Fisher Scientific, New York, NY, USA).
2.7. Statistical Analysis
Quantitative real-time PCR (qRT-PCR) analyses were conducted to assess gene expression levels, utilizing the relative quantification 2−∆∆CT method [28]. A paired-samples t-test was employed to evaluate the susceptibility of T. urticae to short-term heat stress. A significance threshold of p < 0.05 was utilized to determine statistical significance.
3. Results
3.1. Selection and Identification of GST Genes in T. urticae
Based on the comprehensive analysis incorporating whole genome information of T. urticae and transcriptional data following short-term heat shock (39 °C–4 h) compared to a control group (25 °C), we identified six GST genes that exhibited upregulation post short-term heat stress. These genes were thoroughly annotated in NCBI and designated as TuGSTm1, TuGSTm2, TuGSTm3, TuGSTo, TuGSTd1, and TuGSTd2 (Gene entry numbers: XM_015925628.2, XM_015927346.2, XM_015927509.2, XM_015932051.2, XM_015936066.2, and XM_015937313.2) (see Figure 1). Notably, the FPKM (fragments per kilobase of transcript per million mapped reads) values of all six genes significantly increased following short-term heat shock (refer to Figure 2B). Furthermore, upon cloning the coding sequence of these genes, our results demonstrated complete consistency with the sequences archived in NCBI, with no observed base or amino acid mutations. This consistency underscores the reliability and accuracy of our findings.
3.2. Sequence Analysis of Six GST Genes
The characteristics of the six GST genes are summarized in Table 1. The CDS sequence lengths (Open reading frame, ORF) ranged from 642 bp to 732 bp, encoding amino acid (aa) sequences from 192 to 244 residues. The corresponding molecular sizes varied between 22.447 and 27.957 kDa, with theoretical isoelectric points ranging from 4.71 to 6.12. The molecular formulas for each gene are presented in Table 1.
Phylogenetic analysis revealed that the six genes clustered into three distinct GST families: three Mu family GSTs, one Omega family GST, and two Delta family GSTs (see Figure 2A). Notably, TuGSTm2 and TuGSTm3 exhibited the closest evolutionary relationship, while TuGSTd1 and TuGSTd2 also showed a close evolutionary association. This phylogenetic arrangement provides insights into the evolutionary relationships among the GST genes under investigation.
3.3. Structural Characterization of Six GST Genes
Through the prediction of conserved domains, we successfully identified the glutathione (GSH)-binding sites of the GSTs, commonly referred to as G-sites, in all genes except TuGSTo and TuGSTd2. Additionally, we predicted the substrate-binding sites of the GSTs, known as H-sites, across all six genes. Based on predictive software, all G-sites were found to reside within the N-terminal domain, whereas all H-sites were situated within the C-terminal domain (refer to Figure 2C). This distribution pattern sheds light on the structural organization of GST proteins and underscores the functional significance of distinct domains in substrate and cofactor binding.
Each of the six genes was found to possess a single α-helical domain, and the active catalytic sites within the same family exhibited remarkable similarity (refer to Figure 2 and Figure 3). For TuGSTm1, there were eight G-sites (Trp7, Asn8, Asn46, Tyr50, Leu59, Pro60, Ser72, and Lys73) and five H-sites (Asp105, Ser108, Ser109, Ile163, and Gln166). TuGSTm2 displayed eight G-sites (Tyr7, Trp8, Phe46, Lys50, Asn59, Ser60, Gln72, and Lys73) and five H-sites (Glu105, Ala108, Tyr109, Thr162, and Tyr165). In TuGSTm3, there were eight G-sites (Tyr7, Trp8, Trp46, Lys50, Asn59, Leu60, Gln74, and Thr75) and five H-sites (Ile107, Thr110, Leu111, Ile168, and Tyr171). TuGSTo exhibited four H-sites (Gly120, Thr125, Pro126, and Phe181). TuGSTd1 showcased six G-sites (Ser11, His52, Cys53, Val54, Glu66, and Ser67) and nine H-sites (Ser103, Tyr107, Ala108, Asn111, Ala112, Val115, Phe118, Thr163, and Leu166). Lastly, TuGSTd2 presented nine H-sites (Trp101, Gly104, Thr105, Ala108, Ser109, Ala112, Pro116, Ile160, and Thr163). It is important to note that although the G-site of TuGSTo and TuGSTd2 was not directly predicted, it does not necessarily imply its absence.
3.4. Transcriptional Expression of Six GST Genes under Different Heat Stress Conditions
When exposed to 36 °C, the relative expression level of TuGSTm1 was significantly down-regulated after 36 °C for 2 h. With the extension of exposure time, the relative expression level of TuGSTm1 was significantly increased and reached the maximum at 36 °C for 4 h, and then returned to the normal level at 36 °C for 6 h. When exposed to 39 °C, the relative expression levels of TuGSTm1 were significantly up-regulated, rising first, then decreasing, and finally reaching the maximum at 39 °C for 6 h. The relative expression level of the TuGSTm1 gene was significantly up-regulated at 42 °C and increased with the extension of exposure time, reaching the maximum at 6 h (Figure 4A).
When exposed to 36 °C, the relative expression level of TuGSTm2 was significantly up-regulated, reaching the maximum at 36 °C for 2 h, and then showing a trend of first decreasing and then increasing. When exposed to 39 °C, the relative expression level of TuGSTm2 was not significantly different from that of the control group after 39 °C for 2 h, but with the extension of exposure time, the expression level of TuGSTm2 was significantly up-regulated, and the relative expression level reached the maximum at 39 °C for 6 h. When exposed to 42 °C, the relative expression level of TuGSTm2 was significantly up-regulated; it increased with the extension of exposure time, and reached the maximum at 42 °C for 6 h (Figure 4B).
When exposed to 36 °C, the relative expression level of the TuGSTm3 gene was significantly up-regulated and reached the maximum after 36 °C for 2 h. With the extension of exposure time, the relative expression level of the TuGSTm3 gene was significantly down-regulated and reached the lowest level compared with the control group at 36 °C for 6 h. When exposed to 39 °C, the relative expression level of the TuGSTm3 gene was significantly up-regulated. The general trend showed that the relative expression level increased first and then decreased, and the relative expression level reached the maximum at 39 °C for 4 h and the minimum at 39 °C for 6 h. When exposed to 42 °C, the expression level of TuGSTm3 was significantly up-regulated; it increased with the extension of exposure time, and reached the maximum at 6 h (Figure 4C).
When exposed to 36 °C, the relative expression level of TuGSTo was significantly up-regulated and reached the maximum at 36 °C for 2 h, but had no significant difference at 36 °C for 4 h, and was significantly up-regulated again at 36 °C for 6 h. When exposed to 39 °C, the relative expression level of TuGSTo was significantly up-regulated; it increased with the extension of exposure time, and reached the maximum at 6 h. When exposed to 42 °C, the relative expression level of TuGSTo was significantly up-regulated, and the general trend showed that the expression level of TuGSTo increased first, then decreased and then increased again, reaching the maximum at 2 h exposure and the minimum at 4 h exposure (Figure 4D).
When exposed to 36 °C, the relative expression level of TuGSTd1 was significantly down-regulated and reached the minimum at 36 °C for 6 h. At 39 °C, the relative expression level of TuGSTd1 was significantly up-regulated only after exposure at 39 °C for 6 h. At 42 °C, the relative expression levels of TuGSTd1 were significantly up-regulated; they increased with the extension of exposure time, and reached the maximum at 42 °C for 6 h (Figure 4E).
When exposed to 36 °C, the relative expression level of TuGSTd2 was significantly down-regulated, and decreased with the extension of exposure time, reaching the minimum at 36 °C for 6 h. At 39 °C, there was no significant difference in the relative expression level of TuGSTd2 after 2 h exposure at 39 °C compared with the control group, and the relative expression level of TuGSTd2 was significantly down-regulated at 39 °C for 4 h, and only significantly up-regulated and reached the maximum at 39 °C for 6 h. At 42 °C, the relative expression levels of TuGSTd2 were significantly up-regulated; they increased with the extension of exposure time, and reached the maximum at 42 °C for 6 h (Figure 4F).
4. Discussion
T. urticae is a notorious worldwide pest known for its wide range of hosts [29], rapid reproduction rate [30], and strong adaptability to chemical pesticides and adverse environments [31]. Especially in the high-temperature summer season, the reproduction rate is higher and the harm is more serious [32]. As a multifunctional protein, GST plays an indispensable role in vivo [33]. GSTs were initially of concern due to their involvement in insecticide resistance and phytochemical detoxification [34], but in recent years, as more and more GST genes have been identified, other functions have been reported including having antioxidant activities [35], promoting inflammatory responses [36], participating in the regulation of immune responses and cell signaling cascades [37], regulating cholesterol transport and/or metabolism involved in ecdysteroid biosynthesis [38], host feeding adaptive conversion processes [39,40], and odor molecular degradation [41], and participating in insect reproductive physiological processes [42]. The aim of this study was to explore the molecular mechanism of GSTs in response to heat stress in T. urticae.
In this study, we screened three Mu family GSTs, one Omega family GST, and two Delta family GSTs from the transcriptome of T. urticae acquired after heat stress, and preliminarily determined that these six GST genes can reduce the sensitivity of T. urticae to high temperature. According to the whole genome information of T. urticae, there are 31 GSTs in total in T. urticae, all of which belong to cytoplasmic GSTs, including four subfamilies such as Delta, Mu, Omega, and Zeta [25]. According to substrate specificity and immune characteristics, mammalian cytoplasmic GSTs can be divided into Alpha, Mu, Pi, Theta, Zeta, Omega, Sigma, and Kappa families [43]. Higher plant cytoplasmic GSTs can be divided into fourteen families. They are Tau, Phi, Zeta, Theta, TCHQ, Iota (GSTIs), Hemerythrin (GSTHs), DHARs, Lambda (GSTLs), GHRs, mPGES-2s, metaxin, EF1B, and Ure2p [44]. In addition to the four common families of Theta, Zeta, Omega, and Sigma, two unique families of Delta and Epsilon have been found in insects [45]. It is generally believed that proteins whose primary structural sequence similarity is greater than 40% are considered to belong to the same family [43].
The specific GST genes up-regulated during heat stress may vary depending on the organism and its specific physiological and environmental conditions [46]. Different GST isoforms may have distinct substrate specificities and cellular localization [47], allowing them to target different ROS or detoxify specific compounds produced during heat stress. Therefore, we supposed that the reason for the upregulation of these six genes after short-term heat shock is that they share the same specific substrate.
The structure of GSTs in the same family is similar. The closer the phylogenetic relationship is, the more similar the structure is, and the number and location of substrate-binding sites are similar. The phylogenetic analysis of the six GST genes in T. urticae showed that TuGSTm2 and TuGSTm3 had the closest phylogenetic relationship, TuGSTd1 and TuGSTd2 had the closest phylogenetic relationship, and TuGSTo was a separate branch. We predicted the tertiary structure of the six GSTs and the number and location of G-sites and H-sites. Although the amino acids at each active site of GSTs of the same family were different, the locations and number of active sites bound to the substrate were similar. For example, the G-site active sites of TuGSTm1, TuGSTm2, and TuGSTm3 are all in the 7th, 8th, 46th, 50th, 59th, 60th, 72nd, and 73rd places, with eight G-sites and five H-sites. TuGSTd1 and TuGSTd2 each have nine H-sites.
The relative expression levels of the six GST genes increased significantly after exposure to heat stress, especially at 42 °C. Among them, the relative expression levels of TuGSTm1, TuGSTm2, TuGSTd1, and TuGSTd2 reached the highest level after 6 h exposure at 42 °C, and the relative expression levels of TuGSTm3 and TuGSTo reached the highest level after 2 h exposure at 42 °C. We supposed that the potential reason for the disparity between the different recombinant GSTs in the time taken to reach the highest expression is the different affinity for the substrate. Excessive temperatures can cause oxidative stress in organisms [48]. Our previous results showed that short-term heat shock could cause oxidative stress in T. urticae, and the activities of three antioxidant enzymes (SOD, Prx, and CAT) and total antioxidant capacity (T-AOC) were significantly increased [9]. After short-term heat shock, the relative expression levels of the six GST genes were significantly up-regulated, indicating that the six GST genes were involved in clearing excess ROS generated by heat stress and reducing the sensitivity of the T. urticae to high temperature (Figure 5).
5. Conclusions
We used comparative transcriptomics to screen out six GST genes whose relative expression levels were up-regulated after short-term heat shock in T. urticae. We carried out the detailed description and phylogenetic analysis of these six GST genes, predicting the tertiary structure and active sites (G-sites and H-sites) of these six GSTs. The expression patterns of the six GST genes in response to heat stress were determined, and finally, the role of the six GST genes involved in the response to heat stress was determined.
Conceptualization, S.S.; methodology, T.Z. and S.S.; software, T.Z.; validation, T.Z. and S.S.; formal analysis, T.Z.; investigation, T.Z., B.W. and Y.W.; resources, S.S.; data curation, T.Z.; writing—original draft preparation, T.Z.; writing—review and editing, S.S.; visualization, T.Z.; supervision, S.S.; project administration, S.S.; funding acquisition, S.S. All authors have read and agreed to the published version of the manuscript.
Not applicable.
Not applicable.
All data support for this research is included in this article and
The authors declare no conflicts of interest.
Footnotes
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Enlarge this image.Figure 1. Volcanic plot of six GST genes. The final result is based on FPKM as the original data (Figure 2B) (the source data is in Table S3); the Log2FC and p-value were calculated using the DEseq2 method; and data visualization was performed using GraphPad Prism (version 8.0).
Enlarge this image.Figure 2. Characterization of six GST Genes in T. urticae. Six GST genes were identified as belonging to three subfamilies, including three Mu family GSTs, one Omega family GST, and two delta family GSTs. (A) Phylogenetic evolutionary analysis of GST genes. A phylogenetic tree was constructed using MEGA (Version. 11), using the maximum likelihood approach method based on 1000 replicates. (B) Heat map of the response of GST genes to heat stress. C1–4 represent four separate values for the control samples. T1–4 represent four separate values for the treatment samples. RSEM was used to calculate the gene expression levels of each sample. The heatmap function was used for the hierarchical clustering analysis of the six GST genes. The color scale at the bottom denotes the FPKM value from the lowest (blue) to the highest (red). (C) Structural analysis of GST genes. The structural analysis of the GST genes was predicted using NCBI online software Conserved Domain Search Service (CD Search) and visualized using TBtools software. The small vertical icons indicate the amino acids involved in binding sites. The blue icons represent the GSH-binding site of the GSTs (G-site), and the red icons represent the substrate-binding site of the GSTs (H-site).
Enlarge this image.Figure 3. The overall predicted structure of the six GSTs ((A) TuGSTm1; (B) TuGSTm2; (C) TuGSTm3; (D) TuGSTo; (E) TuGSTd1; and (F) TuGSTd2). (a) Visualization of GST structure in cartoon; G-sites are shown with blue stick structure, and H-sites are shown with yellow stick structure. (b) Surface representation of GST genes and the active binding sites are highlighted. Active site residues that are conserved in all GSTs are highlighted in blue and yellow. The blue part indicates the GSH-binding active sites, and the yellow part indicates the substrate-binding sites. (c) All the active sites of GSTs. The amino acid stick structure at the G-sites is shown in blue, and the amino acid stick structure at the H-sites is shown in yellow. The structural model was built using AlphaFold2 and the results were visualized using PYMOL.
Enlarge this image.Figure 3. The overall predicted structure of the six GSTs ((A) TuGSTm1; (B) TuGSTm2; (C) TuGSTm3; (D) TuGSTo; (E) TuGSTd1; and (F) TuGSTd2). (a) Visualization of GST structure in cartoon; G-sites are shown with blue stick structure, and H-sites are shown with yellow stick structure. (b) Surface representation of GST genes and the active binding sites are highlighted. Active site residues that are conserved in all GSTs are highlighted in blue and yellow. The blue part indicates the GSH-binding active sites, and the yellow part indicates the substrate-binding sites. (c) All the active sites of GSTs. The amino acid stick structure at the G-sites is shown in blue, and the amino acid stick structure at the H-sites is shown in yellow. The structural model was built using AlphaFold2 and the results were visualized using PYMOL.
Enlarge this image.Figure 4. Relative expression levels of GST genes ((A) TuGSTm1, (B) TuGSTm2, (C) TuGSTm3, (D) TuGSTo, (E) TuGSTd1, and (F) TuGSTd2) in T. urticae. The statistical difference was determined using the paired samples t-test, data are median, n = 5 biologically independent samples. The measure of variation is the median. The source data is in Table S4.
Enlarge this image.Figure 5. The role of GST in response to short-term heat stress in T. urticae. Two domains on the subunits together constitute the active center of GSTs: an N-terminal GSH-binding domain (G-site) and a C-terminal hydrophobic substrate-binding domain (H-site). The harmful substances produced by oxidative stress caused by heat stress mainly include ROS and lipid peroxides. GPX catalyzes the binding of GST and substrate, completes the metabolism of the substrate, and catalyzes GSH to become GSSH in the case of GSH as an electron donor (GPX: Glutathione peroxidase; GSSH: Glutathione(Oxidized); GSH: Glutathione).
Detailed information of GST genes from T. urticae.
| Gene | ORF | aa | Formula | Molecular Weight (kDa) | Theoretical pI |
|---|---|---|---|---|---|
| TuGSTm1 | 672 | 224 | C1207H1829N303O346S6 | 26.313 | 6.00 |
| TuGSTm2 | 696 | 232 | C1233H1856N302O358S3 | 26.734 | 5.01 |
| TuGSTm3 | 675 | 225 | C1193H1846N292O353S7 | 26.152 | 4.71 |
| TuGSTd1 | 657 | 219 | C1123H1752N280O327S4 | 24.536 | 5.82 |
| TuGSTd2 | 642 | 192 | C1015H1575N259O293S11 | 22.447 | 5.49 |
| TuGSTo | 732 | 244 | C1290H1987N307O365S10 | 27.957 | 6.12 |
Supplementary Materials
The following supporting information can be downloaded at:
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