GmGSTU23 Encoding a Tau Class Glutathione S-Transferase Protein Enhances the Salt Tolerance of Soybean (Glycine max L.)

Salt stress has a detrimental impact on crop yield, quality, and profitability. The tau-like glutathione transferases (GSTs) represent a significant group of enzymes that play a crucial role in plant stress responses, including salt stress. In this study, we identified a tau-like glutathione transferase family gene from soybean named GmGSTU23. Expression pattern analysis revealed that GmGSTU23 was predominantly expressed in the roots and flowers and exhibited a concentration–time-specific pattern in response to salt stress. Transgenic lines were generated and subjected to phenotypic characterization under salt stress. The transgenic lines exhibited increased salt tolerance, root length, and fresh weight compared to the wild type. Antioxidant enzyme activity and malondialdehyde content were subsequently measured, and the data revealed no significant differences between the transgenic and wild-type plants in the absence of salt stress. However, under salt stress, the wild-type plants exhibited significantly lower activities of SOD, POD, and CAT than the three transgenic lines, whereas the activity of APX and the content of MDA showed the opposite trend. We identified changes in glutathione pools and associated enzyme activity to gain insights into the underlying mechanisms of the observed phenotypic differences. Notably, under salt stress, the transgenic Arabidopsis’s GST activity, GR activity, and GSH content were significantly higher than those of the wild type. In summary, our findings suggest that GmGSTU23 mediates the scavenging of reactive oxygen species and glutathione by enhancing the activity of glutathione transferase, thereby conferring enhanced tolerance to salt stress in plants.


Introduction
Soil salinization is widely distributed in more than 60% of countries or regions, causing severe food and economic problems worldwide [1][2][3]. According to statistics, about 7% of the fundamental arable land in China is saline soil and has a trend of increasing yearly [4]. In saline soil, excessive sodium and chloride ions are released into the soil which are actively absorbed by plants, inhibiting seed germination and plant growth and development [5]. Salt stress can seriously affect crops' yield, quality, and benefit, and it has become one of the principal abiotic stresses and limiting factors affecting crop production and agricultural development [5,6]. In order to resist the damage of salt stress, different salt tolerance thaliana. Our study postulated that GmGSTU23 has a crucial role in plant responses to salt stress, ameliorating plant salt tolerance through its regulation of enzyme activity and the proportion of reduced glutathione in plants. The results of this study offer theoretical support in understanding the role of glutathione S-transferase in the molecular mechanisms of plant salt tolerance.

Isolation and Bioinformatics Analysis of the GmGSTU23
Based on the identification of salt-tolerant soybean material resources (unpublished data), a salt-induced gene encoding glutathione S-transferase was isolated from the high salt-tolerant soybean variety Guizao 1 (Table S1). A BLAST search in the soybean reference genome Wm82.a2.v1 identified the gene as Glyma.07G139700 which was named GmGSTU23 and belongs to the GST tau subfamily based on previous studies [59]. The soybean genome database showed that the full-length genomic sequence of GmGSTU23 consisted of two exons and one intron, with a DNA length of 1066 bp, including a 678 bp CDS sequence, which encodes a peptide 226 amino acids in length with an isoelectric point of 5.39 (Table S1, Figure 1A). The amino acid sequence of GmGSTU23 was aligned with the GST members of other plant species. GmGSTU23 shares a degree of homology with members of the GST family from Phaseolus vulgaris, Phaseolus vulgaris, Medicago sativa, Cicer arietinum, and Arabidopsis thaliana, with the highest identity being with PvGST ( Figure 1B). The Pfam database shows that GmGSTU23 has two typical conservative domains called GST-N and GST-C, representing the typical domain characteristics of GST family ( Figure 1A,B). The predictions of the GmGSTU23 protein structural model showed that it has seven protein folds ( Figure 1C). Subcellular localization predictions suggest that expression of GmGSTU23 may be located in the cytoplasm. Phylogenetic analysis revealed that GmGSTU23 homologs of legume species were clustered, and there were several homologous genes in the dicotyledonous plant A. thaliana ( Figure 1D).

Expression Pattern Analysis of the GmGSTU23
In different plant organs, the expression of GmGSTU23 was mainly concentrated in the flower and roots, while the expression in the leaf, stem, and pod was low ( Figure 2A). In the experiment of detecting the response to time, GmGSTU23 showed a significant upward trend after 4 h of NaCl treatment, and the expression level increased significantly with the treatment time and reached the maximum expression at 8 h, and then began to decline ( Figure 2B). In addition, as shown in Figure 2C, the expression of GmGSTU23 in soybean tissues significantly increased with the increase in NaCl concentration after exposure to salt for 8 h.

Expression Pattern Analysis of the GmGSTU23
In different plant organs, the expression of GmGSTU23 was mainly concentra the flower and roots, while the expression in the leaf, stem, and pod was low (Figur In the experiment of detecting the response to time, GmGSTU23 showed a significa ward trend after 4 h of NaCl treatment, and the expression level increased signifi with the treatment time and reached the maximum expression at 8 h, and then be decline ( Figure 2B). In addition, as shown in Figure 2C, the expression of GmGST soybean tissues significantly increased with the increase in NaCl concentration af posure to salt for 8 h.

Generation and Detection of the GmGSTU23 Overexpression Lines
To explore the function of GmGSTU23 in plant salt stress responses, the coding sequence of GmGSTU23 was inserted into the expression vector pTF101 containing a strong CaMV 35S promoter, and the transgenic plant was obtained by the floral dip method. A total of 11 overexpressed lines were selected by herbicide identification and the expression levels of GmGSTU23 in the different lines were determined by qRT-PCR ( Figure S1). Then, three T3 generation transgenic lines (OE-2, OE-5, and OE-11) with high expression levels were selected for studying the salt tolerance phenotype and physiological analysis ( Figure  S1, Figures 3 and 4).

Generation and Detection of the GmGSTU23 Overexpression Lines
To explore the function of GmGSTU23 in plant salt stress responses, the coding sequence of GmGSTU23 was inserted into the expression vector pTF101 containing a strong CaMV 35S promoter, and the transgenic plant was obtained by the floral dip method. A total of 11 overexpressed lines were selected by herbicide identification and the expression levels of GmGSTU23 in the different lines were determined by qRT-PCR ( Figure S1). Then, three T 3 generation transgenic lines (OE-2, OE-5, and OE-11) with high expression levels were selected for studying the salt tolerance phenotype and physiological analysis (Figures S1, 3 and 4).
CaMV 35S promoter, and the transgenic plant was obtained by the floral dip method. A total of 11 overexpressed lines were selected by herbicide identification and the expression levels of GmGSTU23 in the different lines were determined by qRT-PCR ( Figure S1). Then, three T3 generation transgenic lines (OE-2, OE-5, and OE-11) with high expression levels were selected for studying the salt tolerance phenotype and physiological analysis ( Figure  S1, Figures 3 and 4).

Overexpression of GmGSTU23 Confers Phenotypic Tolerance to Salt Stress
To evaluate the salt tolerance of the GmGSTU23 transgenic lines, the salt do sponse phenotype of the seedlings was analyzed in agarose plate and soil cultu shown in Figure 3A, the root elongation of wild-type and transgenic A. thaliana was

Overexpression of GmGSTU23 Confers Phenotypic Tolerance to Salt Stress
To evaluate the salt tolerance of the GmGSTU23 transgenic lines, the salt dose response phenotype of the seedlings was analyzed in agarose plate and soil culture. As shown in Figure 3A, the root elongation of wild-type and transgenic A. thaliana was inhibited to varying degrees under 100 mM or 150 mM NaCl treatment in the plate culture experiment. However, the inhibition of salt stress on the overexpression lines was significantly lower than that in the wild type ( Figure 3B). Specifically, under the treatment of 150 mM NaCl, the root lengths of WT and GmGSTU23 transgenic seedlings were 4.1 cm and 5.2 cm, respectively ( Figure 3B). In the long-term salt stress test in soil culture, 15-day-old plants were continuously irrigated with 200 mM NaCl solution for 20 days. The phenomena of yellowing of plant leaves and stunted growth were revealed under salt stress. It was observed that the growth of wild-type plants was severely affected by salinity, while the growth of the transgenic plants was less affected ( Figure 4A). Compared with the condition without salt stress, the fresh weight of WT and GmGSTU23 transgenic lines under 200 mM NaCl treatment was decreased by 81.2% and 53.1%, respectively ( Figure 4B). In addition, the content of malondialdehyde in the wild-type strain was significantly higher than that in transgenic lines under salt stress, which reflected the degree of membrane peroxidation ( Figure 4B). The data on antioxidant enzyme activities showed that there was no significant difference between transgenic and wild-type plants in the absence of salt stress. Under salt stress, the activities of SOD, POD, and CAT in the wild type were significantly lower than those of the three transgenic lines, while the activity of APX showed the opposite situation ( Figure 4B).

Changes of Glutathione Pool Composition and Related Enzymes under Salt Stress
To analyze the underlying causes of the phenotypic differences, glutathione pools and the associated enzyme activity changes were determined. Under salt stress, GST activity, GR activity, and GSSG content were increased, while GSH content showed the opposite effect ( Figure 5). The analysis between wild-type and transgenic plants showed that the GST activity, GR activity, and GSH content of transgenic Arabidopsis thaliana were significantly higher than those of the wild-type strain under 200 mM NaCl treatment ( Figure 5A,C,D). Specifically, there were significant differences in GST activity and GSH content in the absence of salt stress ( Figure 5A,B). higher than that in transgenic lines under salt stress, which reflected the degree of membrane peroxidation ( Figure 4B). The data on antioxidant enzyme activities showed that there was no significant difference between transgenic and wild-type plants in the absence of salt stress. Under salt stress, the activities of SOD, POD, and CAT in the wild type were significantly lower than those of the three transgenic lines, while the activity of APX showed the opposite situation ( Figure 4B).

Changes of Glutathione Pool Composition and Related Enzymes under Salt Stress
To analyze the underlying causes of the phenotypic differences, glutathione pools and the associated enzyme activity changes were determined. Under salt stress, GST activity, GR activity, and GSSG content were increased, while GSH content showed the opposite effect ( Figure 5). The analysis between wild-type and transgenic plants showed that the GST activity, GR activity, and GSH content of transgenic Arabidopsis thaliana were significantly higher than those of the wild-type strain under 200 mM NaCl treatment (Figure 5A,C,D). Specifically, there were significant differences in GST activity and GSH content in the absence of salt stress ( Figure 5A,B).

Discussion
Salt stress is one of the abiotic stresses that has the most significant impact on agriculture. It can severely reduce the yield and quality of crops by destroying the homeostasis of plant ions, osmosis, and reactive oxygen species [5][6][7]10,11]. During their long evolution, plants have developed many responses to salt stress, such as calcium signals and glutathione-anti-circulating pathways to maintain cell homeostasis and eliminate reactive oxygen species [7,60]. The mechanisms of salt tolerance-related genes have been identified in some species, such as AtHKT1;1, AtHAK5, OsLAX, OsABCB, SOS2, SnRK2, GmDof41, SlWRKY23, etc., and these genes have endowed plants with different degrees of tolerance to salt stress [61][62][63][64][65][66][67]. The ascorbate-glutathione (ASA-GSH) cycle is widespread in living organisms and plays an essential role in plant oxidative defenses and abiotic stress responses [31,68]. During this cycle, GSTs, a family of proteins widely distributed in various organisms, plays a crucial role in detoxification [69]. Specifically, its primary function is to catalyze the coupling of some electrophilic groups with the thiols of reduced glutathione, to change its hydrophobicity so that it can easily cross the cell membrane to the extracellular space after being decomposed, thus achieving detoxification [70,71]. According to previous genome-wide identification reports of the GST family, it has been found that it has functions related to oxidative stress responses to abiotic stress [17,[72][73][74][75]. AtGSTF8, AtGSTU19, and AtGST1 relieved salt stress by maintaining redox homeostasis in A. thaliana roots [76,77]. The glutathione S-transferase gene has been isolated in a study in wild-type soybeans, and transgenic plants showed a higher tolerance to salt at the seedling stage than wild-type plants [43]. Meanwhile, the molecular mechanisms of soybean GST family genes under salt stress have been rarely reported and are unclear.
In this study, a salt-responsive glutathione S-transferase gene, defined as GmGSTU23, was identified as a differentially expressed gene under salt stress. Through sequence alignment and conservative domain analysis, we found that GmGSTU23 shares a high degree of homology with the identified tau subfamily genes, particularly with PaGST ( Figure 1A,D). These results indicated that the GST family genes are highly conserved among different species and may have similar functions in response to environmental stress [17,73]. Analysis of the promoter of GmGSTU23 revealed a total of 3 ABREs (ABAresponsive elements), 2 LTREs (low-temperature responsive elements), 17 MYB-responsive elements, and 20 MYC-responsive element cis-counterparts. Among them, ABRE is mainly involved in the regulation of ABA response or drought expression [78,79]. Based on these results, it was speculated that GmGSTU23 might be associated with abiotic stress responses in plants. Due to the existence of LTREs, some studies have suggested that GmGSTU23 also encodes a heat shock protein in soybeans, which may respond to high-temperature stress, and further studies are needed [80].
The orthologous gene of GmGSTU23 in A. thaliana is At3g09270 with 80% similarity, which is thought to be involved in the early responses of A. thaliana cells to cadmium exposure [19,81]. Although it is similar to the GST family genes of other species in structure such as in the conservative domain, GmGSTU23 may have a unique expression pattern and molecular function (Figures 1 and 2). In some previous studies, the expression patterns of GmGSTU23 were localized in the roots, especially in the root hair [82]. Meanwhile, our results showed that the expression of GmGSTU23 is not confined to the roots, but is expressed in other organs of the plant, especially at high levels in the flowers (Figure 2A), which is similar to the results of some previous reports [83]. After 8h of salt treatment, the expression of GmGSTU23 reached a peak, which suggested that GmGSTU23 might be regulated by some transcription factors and may indirectly participate in plant salt stress responses ( Figure 2B). The 3D structure model of GmGSTU23 did not have a transmembrane domain, and its subcellular localization was predicted to be cytoplasmic. Therefore, it is speculated that GmGSTU23 was expressed in the cytoplasm to catalyze the binding of GSH and ROS (Figures 1 and 2).
In order to further identify the mechanism of GmGSTU23 in enhancing the tolerance of plants to salt stress, we obtained transgenic A. thaliana lines and conducted a salt stress treatment experiment. Compared with the control, the growth of wild-type and transgenic A. thaliana was inhibited under salt stress, and the leaves became withered and yellow (Figures 3A and 4A). The results showed that under salt stress, the root length and fresh weight of transgenic A. thaliana were significantly higher than those of wild type (Figures 3B and 4B). Malondialdehyde is generally considered to be an indicator of the degree of cell membrane peroxidation and the strength of plant responses to adverse conditions [84]. Malondialdehyde content in the transgenic lines was lower than in wild type, indicating that they were less damaged by ROS ( Figure 4B) and that the overexpression of GmGSTU23 enhanced the tolerance of plants to salt stress. The content of antioxidant enzymes in the transgenic A. thaliana and wild-type lines was also determined to study its response mechanism to salt stress ( Figure 4B), and the results were similar to those of previous studies [39][40][41]. Some antioxidant enzymes such as SOD, POD, CAT, and APX can eliminate ROS in cells and reduce the generation of hydrogen peroxide, thereby enhancing salt tolerance [85]. We speculated that the GmGSTU23 transgenic A. thaliana might induce changes in antioxidant enzymes through some pathways to improve the plant's tolerance to salt stress. The measurement of glutathione pool composition and related enzymes showed that the GST activity and GSH content of the transgenic lines were significantly higher than those of the WT strain, regardless of the presence of salt stress ( Figure 5). Therefore, it is speculated that GmGSTU23 directly enhances the activity of glutathione transferase and participates in the clearance of reactive oxygen species with glutathione, thereby enhancing the tolerance of plants to salt stress [31,33,84]. However, little is known about the interactions between GmGSTU23 and other transcription factors, which will need further exploration. Therefore, we will use a yeast library, RNA-Seq, and other techniques to study the function of the GmGSTU23 gene in future studies to increase our understanding of the salt tolerance mechanisms in plants.

Plant Materials and Growth Conditions
The sterilized seeds of soybean variety Guizao 1, obtained from the South China Agricultural University (Guangzhou, China), were selected and sowed in vermiculite. After germination, the seedlings with consistent growth were selected and transferred to a hydroponic system (pH 6.8) with modified Hoagland nutrient solution at 28/25 • C and a 14 h/10 h (light/dark) photoperiod, as described by Ke Wen et al. [86]. The nutrient solution was rejuvenated daily. In the soil culture experiment, the plant organs including the roots, stem, leaf, flower, and pod were collected at the soybean V6 stage and then quickly frozen in liquid nitrogen and stored in an ultra-low-temperature refrigerator [86][87][88]. The experimental plants were grown in the basin with the nutrient soil in an environmentally controlled incubation chamber.

Analysis of Gene Expression Pattern
The plants were processed with the following treatments in the experiment to analyze the gene expression patterns in soybean plants (G. max) [86]. In order to determine the dose-dependent expression patterns, the seedings were cultivated in a solution with 0, 50, 100, 150, and 200 mM NaCl for 8 h. The plant materials were collected after 0, 2, 4, 8, 12, and 24 h in the 200 mM NaCl treatment to determine the time-dependent expression patterns.
To determine organ-specific expression, the plant organs stored in the −80 • C refrigerator described in Section 4.1 were used for qPCR identification [31].
The total RNA was extracted from the plant samples using the RNA-easy Isolation Reagent (Vazyme, Nanjing, China) according to the manufacturer's instructions and then reverse transcribed into cDNA using PrimeScript RT kit (Takara, Shiga, Japan). The gene expression level was evaluated by qPCR using the 2 −∆∆ct method using real-time PCR on a CFX96™ Touch Real-Time PCR System (Bio-Rad, Hercules, CA, USA) with SYBR Premix ExTaq™ II Mix (TaKaRa, Shiga, Japan) [86]. GmACTIN6 (GeneBnak Accession: AAK285830.1) was used as an internal control for three technical replicates. All primers were designed using the NCBI Primer tool (http://www.ncbi.nlm.nih.gov/, accessed on 4 February 2022) and are shown in Table S2.

Cloning and Bioinformatics Analysis of GmGSTU23
In this study, the cDNA of the soybean variety Guizao 1, reverse-transcribed from its mixed tissue RNA sample, was used to clone the gene sequence. GmGSTU23 was extracted with PrimeScript RT Reagent Kit with gDNA eraser (TaKaRa, Shiga, Japan) by PCR using the specific primer pair: 5 -CGCATTCATACGCAGCAATCA-3 and 5 -AGCAATAACTCAACAAGACAAGT-3 . The PCR cloning was performed with primers and cDNA using the following program: 5 min at 94 • C; 35 cycles of 30 s at 94 • C, 1 min at 54 • C, and 2 min at 72 • C; and 5 min at 72 • C for the final extension.
The sequence with the correct molecular size detected by agarose gel electrophoresis was integrated into the pLB vector (Tiangen Biotech, Beijing, China) and sent to a sequencing company (Shenggong, Guangzhou, China) for sequencing. The multiple protein sequence alignment of GmGSTU23 with other glutathione S-transferases (GST) was performed using ClustalW2 (https://www.ebi.ac.uk/Tools/msa/clustalw2/, accessed on 15 January 2023). The tree of gene homology and molecular evolutionary genetics analysis was constructed using MEGA-X software via the Neighbor-Joining (NJ) method. The protein 3D structure of GmGSTU23 was predicted by the phyre2 online prediction website (http: //www.sbg.bio.ic.ac.uk/phyre2/html/page.cgi?id=index, accessed on 17 January 2023). The subcellular localization prediction was performed by the Cell-PLoc 2.0 online tool (http://www.csbio.sjtu.edu.cn/bioinf/Cell-PLoc-2/, accessed on 17 January 2023).

Vector Construction and Transgenic Lines Generation
To obtain transgenic Arabidopsis thaliana lines, the exonic region of GmGSTU23 was amplified using gene-specific primers with XbaI and SacI sites (Table S2). GmGSTU23 was then linearly cloned into the pTF101.1 binary vector with a modified CaMV 35S promoter, which contained a phosphinothricin acetyltransferase (bar) resistance gene (phosphinothricin N-acetyltransferase, PAT) as selection marker for the transgenic line. The constructed vector was transformed into Agrobacterium strain GV3101 by electroporation (Gene Pulser Xcell™ Electroporation Systems, Hercules,CA, USA), and the target gene was transferred into the A. thaliana ecotype Columbia (Col-0) by flower dipping [89]. The T 0 generation of A. thaliana was identified by overexpression by herbicide (Liberty ® , Bayer, Leverkusen, Germany) spraying and PCR molecular identification. Molecular identification of the T 3 transgenic line was performed using the 2 × Taq Plus Master Mix (Vazyme, Nanjing, China) according to the manufacturer's instructions. The specific primers used for PCR were designed based on the vector and the gene sequence (Table S2).

Phenotypes Analysis of GmGSTU23 Transgenic Lines
To analyze the phenotypes of GmGSTU23-overexpressing (OE) and wild-type (WT) A. thaliana under salt stress, three T 3 transgenic lines with high expression levels were selected for 1/2 MS agar plates and soil culture experiments. Specifically, the seeds sterilized with 10% sodium hypochlorite for 10 min were sown on 1/2 MS agar plates and vernalized at 4°C in the dark for four days [86]. Subsequently, the plates were erected and placed in a growth chamber at 22-24 • C, with 60% relative humidity and a 16 h/8 h (light/dark) photoperiod until the roots grew to 1 cm. The seedlings with consistent growth were transferred to 1/2 strength MS agar medium containing different concentrations of NaCl (0 mM, 100 mM, 150 mM) for ten days. The length from the base of the rosette leaf to the top of the straight root was measured with a ruler, and the image was photographed with a Canon EOS 750d camera. In addition, T 3 transgenic seeds and wild-type seeds (Columbia-0) after vernalization were planted in sterile substrate soil (Jiffy, Oslo, Norway) with a 16 h/8 h (light/dark) photoperiod in a GXZ-300D illuminated growth incubator (Jiangnan, Ningbo, China). The two-week-old seedlings were treated with 200 mM NaCl solution irrigation for 20 days. Then, the fresh weight and height of the plants were measured, and the leaf of the plants were quickly frozen in liquid nitrogen and stored in a refrigerator at −80 • C for the determination of various physiological indicators [43].

Statistical Analysis
The experiment was designed with random blocks, and there were at least three experiments and biological repetitions. The data are shown as the mean ± SE (standard error) of the three replicates. For statistical analysis, SPSS Statistics Ver. 22 (IBM, New York, NY, USA) and GraphPad Prism 6 (Boston, MA, USA) were used. One-way ANOVA and Student's t-test were used for comparisons, and p ≤ 0.05 was the threshold for a significant difference.

Conclusions
In this study, a soybean gene GmGSTU23 encoding a tau class glutathione transferase was identified. GmGSTU23 was up-regulated under salt stress. The overexpression of GmGSTU23 in Arabidopsis thaliana lines enhanced their tolerance to salt stress. Based on the physiological data and previous studies, we speculated that GmGSTU23 might improve the activity of glutathione transferase and the clearance of ROS by GSH, thereby alleviating salt toxicity in plants. This study provides some support for understanding the mechanisms of plant regulation of salt tolerance.