PeGSTU58, a Glutathione S-Transferase from Populus euphratica, Enhances Salt and Drought Stress Tolerance in Transgenic Arabidopsis

Glutathione S-transferases (GSTs) play a crucial role in responding to abiotic stress and are an important target for research on plant stress tolerance mechanisms. Populus euphratica is a promising candidate species for investigating the abiotic tolerance mechanisms in woody plants. In our previous study, PeGSTU58 was identified as being associated with seed salinity tolerance. In the present study, PeGSTU58 was cloned from P. euphratica and functionally characterized. PeGSTU58 encodes a Tau class GST and is located in both the cytoplasm and nucleus. Transgenic Arabidopsis overexpressing PeGSTU58 displayed enhanced tolerance to salt and drought stress. Under salt and drought stress, the transgenic plants exhibited significantly higher activities of antioxidant enzymes, including SOD, POD, CAT, and GST, compared to the wild-type (WT) plants. Additionally, the expression levels of several stress-responsive genes, including DREB2A, COR47, RD22, CYP8D11, and SOD1 were upregulated in PeGSTU58 overexpression lines compared to those in WT Arabidopsis under salt and drought stress conditions. Furthermore, yeast one-hybrid assays and luciferase analysis showed that PebHLH35 can directly bind to the promoter region of PeGSTU58 and activate its expression. These results indicated that PeGSTU58 was involved in salt and drought stress tolerances by maintaining ROS homeostasis, and its expression was positively regulated by PebHLH35.


Introduction
Plants are frequently subjected to adverse environmental conditions, such as salt and drought, which limit their growth, development and productivity, ultimately resulting in significant economic losses. Such adverse environmental conditions induce the generation of reactive oxygen species (ROS). Excessive amounts of ROS can cause protein denaturation and mutations in nucleic acids, which can lead to oxidative damage and cell death [1,2]. To prevent damage, plants have evolved highly efficient antioxidant defense mechanisms that enable them to eliminate ROS and protect themselves against oxidative stress. Several ROS-scavenging enzymes have been identified, such as Glutathione S-transferases (GSTs), TRX, and GRX, and their functional role in stress response have has been elucidated [3,4].
The encoding of GSTs is accomplished by a vast gene family and the presence of these genes has been reported in various plant species. Studies have demonstrated that GSTs exhibit multifunctional roles, especially in cellular detoxification [5]. GSTs catalyze reduced glutathione (GSH) which is involved in plant defense system by scavenging free radicals and peroxides. GST proteins contain a GST N-domain which is involved in GSH binding and catalysis, and a GST C-domain involved in hydrophobic substrates binding [6,7]. Based on both protein sequence and structural similarity, plant GSTs can be segregated into eight distinct categories. Among the eight categories, Tau (GSTU) is a plant-specific class of the GST family and possesses a large number of members [8,9].
The role of GST in reducing abiotic stress has been verified in several GSTU genes across various species. In Arabidopsis, the expression of AtGSTU19 is induced by abiotic stress. Overexpression of AtGSTU19 conferred resistance to salt, drought, and methyl viologen stress tolerance [10]. Further studies found that AtGSTU19 mutant showed a higher amount of H 2 O 2 in all root zones and confirmed that AtGSTU19 plays a role in regulating root redox homeostasis and root meristem size [11]. The ectopic expression of rice OsGSTU4 in Arabidopsis demonstrated better growth under salinity and oxidative stresses, achieved by reducing the accumulation of ROS and increasing GST activity in response to such stresses [12,13]. GsGSTU42 and GsGSTU24 from soybean conferred tolerance to flooding by improving the antioxidant capacity [14].
There have been some advances in understanding the regulatory mechanisms of GS-TUs in response to abiotic stress. In transgenic plants overexpressing OsGSTU4, 63 genes showed significantly different expression levels compared to WT plants, with most of these genes being enriched in response to oxidative stress and defense response [13]. Overexpression of AtGSTU19 was observed to enhance the expression of stress-related genes (RD29A, KIN1, and COR15A) [10]. The walnut JrGSTTau1 gene has been shown to regulate stress-related genes, such as NtGST, MnSOD, MAPK9, and CDPK15 [15]. GSTU8 has been found to interact with a calmodulin-like protein involved in regulating cold tolerance [16]. In addition, some transcription factors have been identified as regulators of GST expression. For example, CsWRKY48 was found to activate CsGSTU8 expression by directly binding to the W-box region in its promoter in Camellia sinensis, and overexpression of CsGSTU8 in Arabidopsis improved ROS scavenging capability and increased plant drought resistance [17].
The basic helix-loop-helix (bHLH) family is one of large superfamilies of transcription factors (TFs) in plants. Increasing numbers of bHLH family members have been found to play role in responding to abiotic stresses, such as drought, and salt stress [18,19]. The expression of bHLH35, for example, was notably elevated in response to salt stress in both Lilium pumilum [20]. The AabHLH35 from Anthurium andraeanum increased the tolerance to drought and cold [21]. Ectopic expression of TsPIP1;1 from Thellungiella salsugineae enhanced the salt tolerance in rice by upregulating the expression of bHLH35 [22]. Overexpression of Populus euphratica PebHLH35 increased tolerance to drought stress in transgenic Arabidopsis [23].
Although GSTs play a critical role in protecting herbaceous plants from oxidative stress under stress conditions, the characterization of their function and functional mechanism in perennial woody species remain scarce. P. euphratica, also known as the desert poplar, is mainly distributed in desert regions. Because of its extraordinary adaptation to salt and drought stress, P. euphratica is often used as a candidate species for investigating abiotic tolerance mechanisms in woody plants. In our previous genome-wide association study of P. euphratica seed salinity tolerance, the PeGSTU58 gene was identified as one of the candidate genes [24]. This prompted us to explore its role in salt stress. Thus, PeGSTU58 was isolated and functionally analyzed in the study. PeGSTU58, which is located in the cytoplasm and nucleus, encodes a Tau class GST. Overexpression of PeGSTU58 increased tolerance to salinity and drought stress by improving ROS scavenging in transgenic Arabidopsis. Furthermore, the promoter of PeGSTU58 can be directly bound by PebHLH35, which activates its expression. In summary, these results demonstrate that PeGSTU58 plays a positive role in salt and drought stress resistance.

Isolation and Characterization of PeGSTU58
The coding sequence (CDS) of PeGSTU58 was cloned based on the CCG006870 sequence information (Table S1). The CDS of PeGSTU58 has a length of 660 bp and encodes a protein consisting of 219 amino acids with an expected weight of 25.3 kDa. The alignment of PeGSTU58 with its Arabidopsis orthologs was performed in order to ascertain its conserved domain and it revealed that PeGSTU58 contains a G site, an active site, and an H site ( Figure 1A). In addition, MEGA 6.0 software [25] was utilized to perform a phylogenetic analysis of PeGSTU58 and Tau GST proteins from Arabidopsis, which revealed that PeGSTU58 exhibits a close relationship with AtGSTU27 ( Figure 1B). bound by PebHLH35, which activates its expression. In summary, these results demonstrate that PeGSTU58 plays a positive role in salt and drought stress resistance.

Isolation and Characterization of PeGSTU58
The coding sequence (CDS) of PeGSTU58 was cloned based on the CCG006870 sequence information (Table S1). The CDS of PeGSTU58 has a length of 660 bp and encodes a protein consisting of 219 amino acids with an expected weight of 25.3 kDa. The alignment of PeGSTU58 with its Arabidopsis orthologs was performed in order to ascertain its conserved domain and it revealed that PeGSTU58 contains a G site, an active site, and an H site ( Figure 1A). In addition, MEGA 6.0 software [25] was utilized to perform a phylogenetic analysis of PeGSTU58 and Tau GST proteins from Arabidopsis, which revealed that PeGSTU58 exhibits a close relationship with AtGSTU27 ( Figure 1B).  To determine the subcellular localization of the PeGSTU58 protein, a 35S::PeGSTU58-YFP construct was transiently expressed in tobacco leaf epidermal cells to detect its subcellular localization. After analysis, the fluorescence of the PeGSTU58 was detected in the cytoplasm and nucleus. This was similar to the fluorescent signal of the control YFP ( Figure 2A).
To determine the subcellular localization of the PeGSTU58 protein, a 35S::PeGSTU58-YFP construct was transiently expressed in tobacco leaf epidermal cells to detect its subcellular localization. After analysis, the fluorescence of the PeGSTU58 was detected in the cytoplasm and nucleus. This was similar to the fluorescent signal of the control YFP (Figure 2A). To study the function of a gene, it is important to understand its expression pattern. In this study, the expression pattern of PeGSTU58 in different tissues of P. euphratica, including male flower (MF), female flower (FF), leaf, stem, and root, and under stress conditions was investigated. The results showed that PeGSTU58 was highly expressed in the leaf, followed by FF, while it exhibited low expression in the root ( Figure 2B). Upon NaCl treatment, the expression of PeGSTU58 exhibited an immediate increase, with a nearly three-fold increase observed after only one hour of exposure ( Figure 2C). However, its expression significantly decreased at the 6 h and 12 h marks, reaching only 0.5 times that of the control. Under drought treatment, the expression level of PeGSTU58 was observed to decline, reaching its lowest level, which was only 0.12-fold of the control at 6 h ( Figure  2D). In addition, ARE and TC-rich repeats elements involved in the anaerobic induction, defense and stress responses were detected in the promoter of PeGSTU58 ( Figure S1). To study the function of a gene, it is important to understand its expression pattern. In this study, the expression pattern of PeGSTU58 in different tissues of P. euphratica, including male flower (MF), female flower (FF), leaf, stem, and root, and under stress conditions was investigated. The results showed that PeGSTU58 was highly expressed in the leaf, followed by FF, while it exhibited low expression in the root ( Figure 2B). Upon NaCl treatment, the expression of PeGSTU58 exhibited an immediate increase, with a nearly three-fold increase observed after only one hour of exposure ( Figure 2C). However, its expression significantly decreased at the 6 h and 12 h marks, reaching only 0.5 times that of the control. Under drought treatment, the expression level of PeGSTU58 was observed to decline, reaching its lowest level, which was only 0.12-fold of the control at 6 h ( Figure 2D). In addition, ARE and TC-rich repeats elements involved in the anaerobic induction, defense and stress responses were detected in the promoter of PeGSTU58 ( Figure S1). These results suggest that the expression of PeGSTU58 is regulated by salt and drought stress.

Transgenic PeGSTU58 Arabidopsis Seedling Resistance to Salt and Drought Stress
In our previous study, PeGSTU58 was identified as related to seed salinity tolerance, suggesting its potential role in the salinity response [24]. To investigate the function of PeGSTU58, transgenic Arabidopsis lines with overexpressing PeGSTU58 were generated. Two independent T3 homozygous PeGSTU58-transformed Arabidopsis lines (OE-11 and OE-17) with high transcript levels of PeGSTU58 were used to evaluate the stress tolerance ( Figure 3B).

Transgenic PeGSTU58 Arabidopsis Seedling Resistance to Salt and Drought Stress
In our previous study, PeGSTU58 was identified as related to seed salinity tolerance, suggesting its potential role in the salinity response [24]. To investigate the function of PeGSTU58, transgenic Arabidopsis lines with overexpressing PeGSTU58 were generated. Two independent T3 homozygous PeGSTU58-transformed Arabidopsis lines (OE-11 and OE-17) with high transcript levels of PeGSTU58 were used to evaluate the stress tolerance ( Figure 3B). Two developmental stages of seedling were chosen for stress tolerance analysis. Firstly, seedlings were transferred to 1/2 MS medium containing NaCl and mannitol to compare their growth conditions after one week of growth on 1/2 MS medium. Under normal condition, there were more roots, and branches in PeGSTU58-overexpression seedlings compared to WT. However, under NaCl and drought stress conditions, the growth of PeGSTU58-overexpression seedlings exhibited stronger resistance compared to WT. Specifically, under NaCl stress, the primary root length of WT seedlings was 1.6 cm, and the fresh weight was 2.33 mg, whereas PeGSTU58-overexpression seedlings had a primary root length of 2.8-3.2 cm and a fresh weight of approximately 3.03 mg. Similar results were observed under drought stress ( Figure 3).
Furthermore, two-week-old seedlings were initially grown in soil under normal conditions for two weeks, and were then subjected to either 200 mM NaCl or water withholding to assess their stress tolerance ( Figure 4). After treatment, the growth of WT seedlings was severely inhibited. The PeGSTU58 transgenic plants exhibited a significantly higher relative water content (RWC) of approximately 30% to 40% compared to WT plants which Two developmental stages of seedling were chosen for stress tolerance analysis. Firstly, seedlings were transferred to 1/2 MS medium containing NaCl and mannitol to compare their growth conditions after one week of growth on 1/2 MS medium. Under normal condition, there were more roots, and branches in PeGSTU58-overexpression seedlings compared to WT. However, under NaCl and drought stress conditions, the growth of PeGSTU58-overexpression seedlings exhibited stronger resistance compared to WT. Specifically, under NaCl stress, the primary root length of WT seedlings was 1.6 cm, and the fresh weight was 2.33 mg, whereas PeGSTU58-overexpression seedlings had a primary root length of 2.8-3.2 cm and a fresh weight of approximately 3.03 mg. Similar results were observed under drought stress ( Figure 3).
Furthermore, two-week-old seedlings were initially grown in soil under normal conditions for two weeks, and were then subjected to either 200 mM NaCl or water withholding to assess their stress tolerance ( Figure 4). After treatment, the growth of WT seedlings was severely inhibited. The PeGSTU58 transgenic plants exhibited a significantly higher relative water content (RWC) of approximately 30% to 40% compared to WT plants which had an RWC of approximately 20% ( Figure 4B). Relative electrical conductivity (REC) can be used to characterize the damage degree of plant cell membrane. Furthermore, the PeGSTU58 transgenic plants showed an REC of around 30%, lower than that of the WT plants, which had an REC of approximately 50% under salt and drought stress ( Figure 4C). These results indicate that overexpression of PeGSTU58 alleviates growth inhibition of seedlings under salt and drought treatment.
had an RWC of approximately 20% ( Figure 4B). Relative electrical conductivity (REC) can be used to characterize the damage degree of plant cell membrane. Furthermore, the PeGSTU58 transgenic plants showed an REC of around 30%, lower than that of the WT plants, which had an REC of approximately 50% under salt and drought stress ( Figure  4C). These results indicate that overexpression of PeGSTU58 alleviates growth inhibition of seedlings under salt and drought treatment. PeGSTU58-overexpression and the WT seedlings were cultivated under normal growth conditions for two weeks in soil. Water was withheld for 10 days. This was followed by a rehydration period to examine the plants' ability to withstand drought stress. For salt treatment, 30 mL of 200 mM NaCl was irrigated to the seedlings every 3 days for 3 weeks. Photographs were taken after each course of treatment. (B,C) The relative water content (RWC) and relative electrical conductivity (REC) of WT and the PeGSTU58-overexpression seedlings under control, salt, and drought stress was measured. The values presented herein result from an average calculation based on three separate replicates. Significance test was conducted using Student's t-test (** p < 0.01).

PeGSTU58 Mediates ROS Scavenging Capability
Abiotic stress can induce the production of ROS, including H2O2 and O 2− [26]. In order to assess the ROS content, 3, 3′-diaminobenzidine (DAB) and Nitroblue tetrazolium (NBT) stainings were used to measure the levels of H2O2 and O 2− , respectively. Under normal growth conditions, there was no discernible difference in DAB or NBT staining between PeGSTU58 transgenic and WT plants ( Figure 5A). After salt and drought treatment, the staining signals in WT plants were darker than in the PeGSTU58-overexpression seedlings ( Figure 5A  (A) PeGSTU58-overexpression and the WT seedlings were cultivated under normal growth conditions for two weeks in soil. Water was withheld for 10 days. This was followed by a rehydration period to examine the plants' ability to withstand drought stress. For salt treatment, 30 mL of 200 mM NaCl was irrigated to the seedlings every 3 days for 3 weeks. Photographs were taken after each course of treatment. (B,C) The relative water content (RWC) and relative electrical conductivity (REC) of WT and the PeGSTU58-overexpression seedlings under control, salt, and drought stress was measured. The values presented herein result from an average calculation based on three separate replicates. Significance test was conducted using Student's t-test (** p < 0.01).

PeGSTU58 Mediates ROS Scavenging Capability
Abiotic stress can induce the production of ROS, including H 2 O 2 and O 2− [26]. In order to assess the ROS content, 3, 3 -diaminobenzidine (DAB) and Nitroblue tetrazolium (NBT) stainings were used to measure the levels of H 2 O 2 and O 2− , respectively. Under normal growth conditions, there was no discernible difference in DAB or NBT staining between PeGSTU58 transgenic and WT plants ( Figure 5A). After salt and drought treatment, the staining signals in WT plants were darker than in the PeGSTU58-overexpression seedlings ( Figure 5A), implying that there was a lower level of H 2 O 2 and O 2− in the transgenic plants than in the WT plants under stress treatment.
Antioxidant enzymes play a crucial role in alleviating excess ROS. To investigate the mechanism of lower levels of H 2 O 2 and O 2− in the transgenic plants, the activities of GST, superoxide dismutase (SOD), peroxidase (POD), and catalase (CAT) in PeGSTU58overexpression seedlings and WT seedlings were measured under both normal and stress conditions. The results indicated that the transgenic plants displayed higher GST activity than WT plants. Moreover, the discrepancy in GST activity was found to have amplified after applying the salt and drought treatment ( Figure 5B). Furthermore, while there was no significant disparity in the levels of POD, SOD, and CAT activities between PeGSTU58overexpressing seedlings and WT seedlings under normal circumstances, under stressful conditions, the transgenic seedlings exhibited higher levels of POD, SOD, and CAT than that of the WT (Figure 5C,E). These findings suggest that overexpression of PeGSTU58 can improve the tolerance to salt and drought by activating the antioxidant system. Antioxidant enzymes play a crucial role in alleviating excess ROS. To investigate the mechanism of lower levels of H2O2 and O 2− in the transgenic plants, the activities of GST, superoxide dismutase (SOD), peroxidase (POD), and catalase (CAT) in PeGSTU58-overexpression seedlings and WT seedlings were measured under both normal and stress conditions. The results indicated that the transgenic plants displayed higher GST activity than WT plants. Moreover, the discrepancy in GST activity was found to have amplified after applying the salt and drought treatment ( Figure 5B). Furthermore, while there was no significant disparity in the levels of POD, SOD, and CAT activities between PeGSTU58overexpressing seedlings and WT seedlings under normal circumstances, under stressful conditions, the transgenic seedlings exhibited higher levels of POD, SOD, and CAT than that of the WT (Figure 5C,E). These findings suggest that overexpression of PeGSTU58 can improve the tolerance to salt and drought by activating the antioxidant system.

The Expression of Stress-Related Genes was Regulated in Transgenic PeGSTU58 Plants
To clarify the functional mechanism of PeGSTU58 in response to salt and drought stress, we examined the expression of stress-related genes previously reported to be upregulated in plants overexpressing GSTU [11]. These genes included DREB2A, COR47, RD22, CYP8D11, and SOD1, and their expression levels were analyzed in the leaves of both WT and the mixture of two transgenic lines under normal and stress conditions using qRT-PCR ( Figure 6).

The Expression of Stress-Related Genes Was Regulated in Transgenic PeGSTU58 Plants
To clarify the functional mechanism of PeGSTU58 in response to salt and drought stress, we examined the expression of stress-related genes previously reported to be upregulated in plants overexpressing GSTU [11]. These genes included DREB2A, COR47, RD22, CYP8D11, and SOD1, and their expression levels were analyzed in the leaves of both WT and the mixture of two transgenic lines under normal and stress conditions using qRT-PCR ( Figure 6).
Under normal conditions, the expression level of CYP8D11 was higher in PeGSTU58overexpression seedlings than in WT, and the other genes showed no any significant difference. However, in the presence of salt and drought stress, these genes exhibited an upregulation in their expression levels ( Figure 6). Notably, the expression levels were significantly higher in PeGSTU58-overexpression seedlings than in WT. Taken together, these results suggest that the expression of stress-related genes under salt and drought stress conditions was significantly enhanced in transgenic plants overexpressing PeGSTU58.

PebHLH35 Binds to the PeGSTU58 Promoter and Activates Its Expression
To further investigate the regulatory mechanism of PeGSTU58, PlantPAN v.3.0 [27] was used to predict the upstream regulators of PeGSTU58. The analysis suggested that putative bHLH proteins may combine with the promoter PeGSTU58. Among the bHLH proteins, PebHLH35 was confirmed to play roles in salt tolerance in a previous study [23] and it was predicated that PebHLH35 might bind to the "TCAACTTGA" motif in the promoter region of PeGSTU58. To confirm this, we generated a construct containing the promoter fragment of PeGSTU58 with the "TCAACTTGA" motif (pHis2-PeGSTU58 pro ), without the "TCAACTTGA" motif (pHis2-PeGSTU58 ∆pro ) and used them for yeast one-hybrid (Y1H) assay with pGADT7-Rec2-PebHLH35. The p53-His2 and pGADT7-p53 were used as positive controls and p53-His2 and pGADT7-Rec2-PebHLH35 were used as negative controls. All the yeast strains exhibited robust growth in a synthetic-defined (SD) medium (/-Trp/-Leu). The co-transformed yeast strains carrying pHis2-PeGSTU58 pro and pGADT7-Rec2-PebHLH35 were able to grow on the SD medium (/-Trp/-Leu/-His) containing 50 mm 3-AT ( Figure 7C). In contrast, no growth was observed in the negative control group and the combination of pHis2-PeGSTU58 ∆pro and pGADT7-Rec2-PebHLH35 on the SD medium (/-Trp/-Leu/-His) containing 50 mm 3-AT ( Figure 7C). These results indicate that PebHLH35 can bind to the promoter of PeGSTU58 containing the "TCAACTTGA" motif. Under normal conditions, the expression level of CYP8D11 was higher in PeGSTU58overexpression seedlings than in WT, and the other genes showed no any significant difference. However, in the presence of salt and drought stress, these genes exhibited an upregulation in their expression levels ( Figure 6). Notably, the expression levels were significantly higher in PeGSTU58-overexpression seedlings than in WT. Taken together, these results suggest that the expression of stress-related genes under salt and drought stress conditions was significantly enhanced in transgenic plants overexpressing PeGSTU58.

PebHLH35 Binds to the PeGSTU58 Promoter and Activates its Expression
To further investigate the regulatory mechanism of PeGSTU58, PlantPAN v.3.0 [27] was used to predict the upstream regulators of PeGSTU58. The analysis suggested that putative bHLH proteins may combine with the promoter PeGSTU58. Among the bHLH proteins, PebHLH35 was confirmed to play roles in salt tolerance in a previous study [23] and it was predicated that PebHLH35 might bind to the "TCAACTTGA" motif in the promoter region of PeGSTU58. To confirm this, we generated a construct containing the promoter fragment of PeGSTU58 with the "TCAACTTGA" motif (pHis2-PeGSTU58pro), without the "TCAACTTGA" motif (pHis2-PeGSTU58∆pro) and used them for yeast one-hybrid (Y1H) assay with pGADT7-Rec2-PebHLH35. The p53-His2 and pGADT7-p53 were used as positive controls and p53-His2 and pGADT7-Rec2-PebHLH35 were used as negative controls. All the yeast strains exhibited robust growth in a synthetic-defined (SD) medium (/-Trp/-Leu). The co-transformed yeast strains carrying pHis2-PeGSTU58pro and pGADT7- To further verify the suppression or activation of PeGSTU58 by PebHLH35, a luciferase (LUC) assay was carried out for the transient expression in tobacco leaves. The leaves were co-infiltrated with 35S::PebHLH35 and PeGSTU58pro::LUC constructs ( Figure 7D). Strong fluorescent signals were observed in the tobacco leaves that were co-infiltrated with both constructs (Figure 7E), while no fluorescence was observed in the control samples. These results demonstrate that PebHLH35 is capable of directly binding to the PeGSTU58 promoter and activating its expression. To further verify the suppression or activation of PeGSTU58 by PebHLH35, a luciferase (LUC) assay was carried out for the transient expression in tobacco leaves. The leaves were co-infiltrated with 35S::PebHLH35 and PeGSTU58pro::LUC constructs ( Figure 7D).

Discussion
GSTs are a diverse and large family of enzymes that play a pivotal role in plant stress tolerance by protecting against oxidative damage [26]. The functions of GSTs have been extensively studied in various plant species, including Arabidopsis [28], rice [29], Camellia sinensis [17], barley [30], potato [31], pepper [32], Vitis vinifera [33], Tamarix hispida [34], maize [35], and Citrus sinensis [36]. In P. euphratica, only PeGSTF4 has been investigated for its positive role in stress tolerance [37]. Based on our previous study [24], PeGSTU58 was identified as a candidate gene involved in salinity tolerance in P. euphratica, and its regulatory function in response to abiotic stress was investigated in this study.
Phylogenetic analysis indicated that PeGSTU58 is closely related to AtGSTU27 and belongs to the Tau class (Figure 1), suggesting that PeGSTU58 may have conserved roles with the members of the Tau class and may be involved in abiotic stress responses. Protein structure analysis showed that PeGSTU58 has a conserved GSH binding site ( Figure 1A), which plays a crucial role in the conjugation of the GSH moiety, implying that PeGSTU58 may have the ability to catalyze GSH. This speculation was confirmed by the higher GST activity observed in PeGSTU58-overexpression plants compared to WT plants under normal and drought stress conditions ( Figure 5A).
Gaining insight into the subcellular localization of proteins is advantageous for the functional analysis of genes. MdGSTU12 from Malus domestica was found to be located in the cytoplasm where it functions as a cytoplasmic transporter and contributes to the accumulation of anthocyanin [38]. OsGSTU4 was observed in both the cytoplasm and nucleus where it enhances the expression of genes involved in stress responses and cellular detoxification processes [12]. The dual localization of PeGSTU58 in the cytoplasm and nucleus ( Figure 2A) suggests that it may have multiple roles in cellular processes. In addition to its potential function as a transporter within the cytoplasm, the nuclear localization of PeGSTU58 implies that it may play a role in regulating transcription. Additionally, the ability of PeGSTU58 to regulate the expression of DREB2A and RD22 genes in PeGSTU58overexpression plants ( Figure 6) suggests that it may function as a transcriptional regulator. However, the specific molecular mechanisms through which PeGSTU58 controls gene expression require further investigation. Moreover, further studies are necessary to identify the interacting partners and target genes of PeGSTU58 in both cytoplasmic and nuclear compartments to fully understand its biological functions.
PeGSTU58-overexpression transgenic plants were employed to evaluate the function of PeGSTU58. The transgenic plants showed superior growth under salt and drought conditions, as evidenced by their increased root length and fresh weight, compared to WT plants. Plant stress leads to ROS accumulation. Excess ROS is then eliminated by the enzymatic defense systems. In this study, the activities of POD, SOD, and CAT increased and the accumulation of ROS was reduced when PeGSTU58-overexpressed plants were exposed to salt and drought stress, suggesting a positive role of PeGSTU58 in ROS scavenging. These results are consistent with previous research on the functional properties of GSTUs [12][13][14] and indicate that GSTUs have a crucial role in plant stress responses by enhancing the activities of antioxidant enzymes.
Under abiotic stress, GST expression can be consistently altered, with some GSTs exhibiting reduced expression. For example, in T. hispida, the expression of ThGSTZ1 was significantly reduced under salt and drought stress, and its overexpression enhanced the plant's ability to withstand these stresses [34]. In another study, the expression of GsGSTU13 was induced after exposure to NaCl for 1 h, but its expression was later inhibited from 3 to 12 h [39]. However, many GST genes activated by abiotic stress have been investigated and found to play a positive role in stress response [11,12,17]. In our study, we found that the expression of PeGSTU58 was induced in the early stage of salt treatment, but inhibited in the later stage under drought. The findings imply that the GST superfamily's transcript regulation is influenced by numerous different mechanisms.
In the process of signal transduction in response to abiotic stress, various transcription factors (TFs), such as ERF [29] and WRKY [17], bind to the promoters of GSTs. This binding ultimately leads to the modulation of GST expression, which enables an organism to effectively respond to the external environment. In addition, one GST also can be regulated by multiple TFs. For example, in Juglans regia, JrGSTTau1 can be activated by JrMYC2, JrDof1, and JrWRKY7 in response to osmotic stress [15]. In response to oxidative stress and extreme temperature, AtERF2 positively modulates the expression of AtGST11, whereas homeobox protein 6 exerts a negative regulatory effect [40]. In our study, we detected several cis-elements in the promoter of PeGSTU58 ( Figure S1) and predicted TFs that may regulate its expression. PebHLH35 was confirmed to be directly bind to the promoter of PeGSTU58 using yeast one-hybrid assays and luciferase analysis. Previous study has shown that increased expression of PebHLH35 leads to improved resistance against water-deficit stress [23]. Therefore, we propose that PeGSTU58 acts as a positive factor in abiotic stress tolerance, at least in part, being regulated by PebHLH35.
In summary, PeGSTU58 was isolated and characterized from P. euphratica as a positive regulator of salt and drought stress. Overexpression of PeGSTU58 resulted in significant enhancement of salt and drought tolerance in plants by reducing the levels of ROS and elevating the activity of antioxidant enzymes. Furthermore, PeGSTU58 overexpression was found to induce the expression of genes associated with stress response. In addition, PebHLH35 was identified as a potential upstream regulator of PeGSTU58. The investigation provides a foundation for gaining deeper insights into the function of PeGSTU58 and the underlying molecular mechanism of salt and drought tolerance in P. euphratica.

Plant Material and Treatments
A. thaliana (ecotype: Col-0) was selected as wild-type (WT) and used as a transgene receptor. The Arabidopsis seeds underwent a sterilization procedure that involved two rounds of exposure to 75% ethanol and a subsequent single exposure to 100% ethanol. After the sterilized seeds were dry, they were sown in a plate containing 1/2 MS medium and subsequently stored at 4 • C for 3 days. The plate were cultivated under 20-22 • C, while being exposed to a light and dark cycle of 16 h and 8 h, respectively. Two weeks after germination, the seedlings were transplanted into soil for further cultivation.

A. thaliana Transformation and Isolation of Transformed Plants
The CDS of PeGSTU58 was cloned and constructed into over-expression vector PMDC32, which was driven by a 35S promoter, labelled as 35S::PeGSTU58. The floral dip method was employed for Agrobacterium-mediated A. thaliana transformation [41]. Upon screening the T0 seeds in a selection medium containing 25 mg/L hygromycin, over 30 transgenic lines were successfully procured. After qRT-PCR analysis, two independent transgenic lines with high abundance of PeGSTU58 were used for further study.

RNA Extraction and qRT-PCR Assays
The RNA was isolated using the RNeasy Plant kit (Tiangen, Beijing, China) and qRT-PCR were carried out in accordance with their protocol. AtActin7 was chosen as a reference gene for gene expression analysis in A. thaliana. PeActin, PeEF1α, and PeHIS were selected as reference genes for different tissue types, NaCl treatment, and drought treatment in P. euphratica [42]. For the qRT-PCR analysis, each sample was subjected to three biological replicates and four technical replicates. The primers were listed in Table S2.

Subcellular Localization of PeGSTU58
To create the 35S::PeGSTU58-YFP construct, the lack of a stop codon in the CDS of PeGSTU58 was incorporated into the pEarleyGate101 vector. This construct was then transformed into Agrobacteriu. After infiltration of Agrobacterium to tobacco leaves for 2 days, the fluorescence signal was observed using a LSM 510 confocal laser scanning microscope.

Stress Tolerance Analysis
For the salt and drought tolerance experiments, one-week-old seedlings of 35S::PeGSTU58 and WT were cultivated on 1/2 MS medium containing 150 mM NaCl or 200 mM mannitol. The fresh weight and primary root length were assessed after a treatment period of one week.
In addition, two-week-old seedlings of 35S::PeGSTU58 and WT grown on normal medium for two weeks, were planted in soil. After they were grown in soil for four weeks under normal conditions, the seedlings were subjected to salt treatment by administering 30 mL of 200 mM NaCl through irrigation. After two weeks of salt treatment, the seedlings were irrigated with pure water for seven days. For drought treatment, the seedlings were grown in well-watered soil for two weeks. Irrigation was then withheld until the seedlings began to wilt. After this, they were well watered for a further week.

Physiological Measurements and Histochemical Assays
Fresh leaves of 35S::PeGSTU58 and WT under normal, drought, and salt stress condition were collected for physiological measurement including REC, RWC, and enzyme activities. The REC and RWC were measured according to a previous method [43]. The activities of antioxidant enzymes (SOD, POD, CAT, and GST) were measured using the test kit (Solarbio, Beijing, China) in accordance with protocols. Three replicates of each index measurement were taken.
For DAB staining, the DAB staining solution with 1 mg/mL concentration was adjusted to pH 3.8 with dilute hydrochloric acid. The obtained leaves were placed in the DAB staining solution and evacuated in a vacuum pump for 20 min. They were then placed in a dark place at 25 • C for 6-8 h [44]. For NBT staining, the NBT staining solution with 0.5 mg/mL concentration was adjusted to pH 7.8. The obtained leaves were placed in the NBT staining solution and vacuumed for 20 min, then placed in a dark place at 25 • C for 1 h [45]. After staining, the leaves were decolorized in a 95 • C water bath in 95% anhydrous ethanol. Lastly, images were captured using a Zeiss Axio Imager A1 microscope (Carl Zeiss, Jena, Germany).

Prediction the Upstream Regulators of PeGSTU58
The promoter sequence of PeGSTU58 was uploaded to the PlantPAN v.3.0 (http: //plantpan.itps.ncku.edu.tw/promoter.php accessed on 12 December 2021) online software to predict the upstream regulators of PeGSTU58, according to the previous description [27].

Yeast One-Hybrid (Y1H) Assay
The promoter fragment of PeGSTU58 and the full-length CDS of PebHLH35 were inserted into pGADT7-Rec2 and pHIS2 vector to generate the recombinant plasmid pHis2-PeGSTU58 pro and pGADT7-Rec2-PebHLH35, respectively. The plasmid pHis2-PeGSTU58 pro and pGADT7-Rec2-PebHLH35 were co-transformed into Y187 yeast strain and screened on the SD/-Leu/-Trp deficient medium. The successfully transformed yeast strains were proliferated and cultured to an OD value of 0.8, and then diluted 10, 100, and 1000 times and dripped onto the SD/-Leu/-Trp/-His containing 50 mM 3-AT, respectively.

Transient Dual-Luciferase Assays
The luciferase reporter construct (PeGSTU58pro::Luc) was generated by inserting the promoter fragment of PeGSTU58 into the pGreenII 0800-LUC vector. The effector vector (35S::PebHLH35) was produced by incorporating PebHLH35 into the pCAMBIA 1302 vector. The vectors were then transformed into Agrobacterium and were subsequently co-expressed in tobacco leaves in a transient manner. After being cultured in an incubator under control conditions for 48 h, the relative LUC activity levels fluorescent were measured and images were captured using plant view 100 (BLT, Xi'an, China).

Statistical Analyses
Statistical analyses were conducted using SPSS statistics 19.0 (SPSS Inc., Chicago, IL, USA). The data underwent a comparison process using t test. In the event that the p value was below 0.05, the variances were regarded as significant.