Genome-Wide Identification of GSTs Gene Family and Functional Analysis of BraGSTF2 of Winter Rapeseed (Brassica rapa L.) under Cold Stress

The largest gene families in plants were found to be Glutathione transferases (GSTs), which played significant roles in regulating plant growth, development, and stress response. Within the GSTs gene family, members were found to play a crucial role in the low-temperature response process of plants. A comprehensive study identified a total of 70 BraGSTs genes. Cluster analysis results demonstrated that the BraGSTs in Brassica rapa (B. rapa) could be categorized into eight sub-families and were unevenly distributed across ten chromosomes. The 39 BraGSTs genes were found to be organized into 15 tandem gene clusters, with the promoters containing multiple cis-elements associated with low-temperature response. Cold stress was observed to stimulate the expression of 15 genes, with the BraGSTF2 gene exhibiting the highest level of expression, suggesting its significant involvement in winter B. rapa’s response to low-temperature stress. Subcellular localization analysis of the BraGSTF2 protein indicated its potential expression in both the cell membrane and nucleus. The analysis of stress resistance in BraGSTF2 transgenic Arabidopsis thaliana lines demonstrated that the over-expression of this gene resulted in significantly elevated levels of SOD, POD activity, and SP content compared to the wild type following exposure to low temperatures. These levels reached their peak after 24 h of treatment. Conversely, the MDA content was lower in the transgenic plants compared to the wild-type (WT) Arabidopsis (Arabidopsis thaliana L.). Additionally, the survival rate of BraGSTF2 transgenic Arabidopsis was higher than that of the WT Arabidopsis thaliana, suggesting that the BraGSTF2 gene may play a crucial role in enhancing the cold stress tolerance of winter B. rapa. This study lays a foundation for further research on the role of the BraGSTs gene in the molecular regulation of cold resistance in winter B. rapa.


Introduction
Winter rapeseed (Brassica rapa L.), a type of rapeseed that can withstand winter conditions in northern China, plays a crucial role in ensuring a stable supply of edible oil and improving economic benefits [1,2].The cold-resistant variety known as Longyou-7 has demonstrated its ability to survive extreme temperatures as low as −30 • C, indicating the presence of numerous cold-resistance genes within it [3,4].Consequently, Longyou-7 serves as a valuable resource for investigating and understanding the mechanisms underlying cold resistance in winter Brassica rapa (B.rapa).
Cold stress has been found to have a significant impact on various aspects of plant physiology, biochemistry, metabolism, and molecules, ultimately affecting the growth and development of plants [5,6].Specifically, the activities of antioxidant enzymes, including superoxide dismutase, glutathione transferase, glutathione reductase, glutathione peroxidase, Genes 2023, 14, 1689 2 of 16 ascorbate peroxidase, and catalase, have been observed to increase under cold stress.Additionally, the involvement of non-enzymatic antioxidants, such as tripeptide mercaptan and ascorbic acid, has been shown to directly contribute to the stability of cell membranes [7][8][9][10][11][12].It is worth noting that glutathione transferase (GSTs) is a highly conserved and widely distributed protein family with multifunctional properties, suggesting its ancient origins and broad significance in various biological processes [13].
The GSTs, a multifunctional protein family, have been identified in numerous plant species, with 55 genes found in Arabidopsis [14], 179 in Brassica napus [15], 84 in Hordeum vulgare [16], 59 in Gossypium raimondii [17], 75 in Brassica rapa [18], 65 in Brassica oleracea [19], and 23 in Citrus sinensis [20].These GSTs genes play a crucial role in plant secondary metabolism, growth, and development, particularly in response to biotic and abiotic stress [21,22].For instance, the AtGSTU17 gene in Arabidopsis regulates seed development, including hypocotyl elongation and anthocyanin accumulation [16].Additionally, the transfer of the VvGSTF13 gene enhances Grape's salt and drought tolerance [23].The ThGSTZ1 gene has been found to have a dual role in enhancing tolerance to drought and salt stress, as well as improving antioxidant activity through the regulation of reactive oxygen species (ROS) metabolism in plants [24,25].GSTs genes have been extensively studied for their involvement in gene expression related to salt stress tolerance [26].
While there is substantial research on the impact of GSTs genes on oxidative and temperature stress tolerance, limited research has been conducted on their role in cold resistance in winter B. rapa.This study aims to comprehensively analyze the expression and function of GSTs genes, including GSTF2, under low-temperature stress conditions.The purpose of this study was to conduct a comprehensive analysis of the number of GSTs gene families and the function of the BraGSTF2 gene under low-temperature stress in winter B. rapa at the whole genome level.This research aimed to establish a theoretical foundation for analyzing the regulatory mechanism of the gene family under cold stress and abiotic stress in winter B. rapa.

Identification and Protein Characterization of BraGSTs Gene Family
The present study focused on the identification and characterization of the BraGSTs gene family in B. rapa.A comprehensive search was conducted using specific keywords and the Pfam number in the B. rapa database.These proteins were identified as having the reported peroxidase domains after sequence analysis using SMART, CDD, and Pfam.As a result, a total of 70 members belonging to the BraGSTs gene family were successfully identified.Additionally, the members of the BraGSTs gene family were named based on their respective chromosomal positions.Furthermore, an analysis of the physical and chemical properties of all BraGSTs proteins was conducted.This analysis revealed that the number of amino acids in these proteins ranged from 114aa to 600aa.Moreover, the theoretical isoelectric point (PI) of BraGSTs proteins varied from 4.84 to 9.54.The stability index exhibited a range of values spanning from 27.07 to 95.27.Similarly, the fat index displayed a range from 70.12 to 106.00.The hydrophilic and hydrophobic range encompassed values ranging from −0.52 to 0.16.The primary constituents of the BraGSTs protein were identified as carbon (C), hydrogen (H), nitrogen (N), oxygen (O), and sulfur (S).The analysis of the protein's secondary structure prediction revealed that the BraGSTs proteins predominantly consisted of α helices, β sheets, random coils, and extended chains (Supplementary Table S1).

Phylogenetic Analysis, Chromosome Distribution, and Gene Duplication in BraGSTs Gene Family
The NJ method was used to construct a cluster map that divided 70 GSTs genes into 8 main subfamilies in B. rapa (Figure 1).These subfamilies include the -subunit of translation elongation factor (EF1G), Phi (GSTF), Tau (GUTF), Zeta (GSTZ), DHAR, Lambda (GSTL), Theta (GSTT), and TCHQD.Among these subfamilies, the 18 BraGSTs genes were classified as Phi (GSTF), the 39 BraGSTs genes as Tau (GSTU), the four BraGSTs genes as DHAR, and one BraGSTs gene as TCHQD.Moreover, the Zeta, Lambda, Theta, and EFIG subfamilies each consisted of two distinct BraGSTs genes(Table 1).Based on the distribution map of the BraGSTs gene family on chromosomes in B. rapa, as depicted in Figure 2, it is evident that the genes were dispersed across ten distinct chromosomes.Notably, Chromosome 7 exhibits the highest number of gene distributions, with 14 BraGSTs genes, while Chromosome 1 harbors only one BraGSTs gene.In comparison to other subfamilies, the Tau family members display a wide distribution pa ern, spanning nine chromosomes (A01, A02, A03, A04, A05, A06, A07, A08, A09).Based on the distribution map of the BraGSTs gene family on chromosomes in B. rapa, as depicted in Figure 2, it is evident that the genes were dispersed across ten distinct chromosomes.Notably, Chromosome 7 exhibits the highest number of gene distributions, with 14 BraGSTs genes, while Chromosome 1 harbors only one BraGSTs gene.In comparison to other subfamilies, the Tau family members display a wide distribution pattern, spanning nine chromosomes (A01, A02, A03, A04, A05, A06, A07, A08, A09).
Tandem repeats play an important role in expanding the gene family during the evolutionary process.In this study, a total of 70 BraGSTs genes were subjected to sequence alignment in order to identify genes with tandem repeats.The analysis revealed that 38 genes (55.7%) formed 15 tandem repeat clusters.Among these clusters, the A09 chromosomes exhibited the highest number of tandem repeat clusters, with a total of four clusters.Additionally, the A02, A03, A06, and A07 chromosomes each contained two tandem repeat clusters.On the other hand, the A04, A05, and A10 chromosomes harbored only one tandem repeat cluster each, while no tandem repeats were observed on the A01 and A08 chromosomes.This implies that tandem repeats have a significant impact on the proliferation of the BraGSTs gene family.Tandem repeats play an important role in expanding the gene family during the evolutionary process.In this study, a total of 70 BraGSTs genes were subjected to sequence alignment in order to identify genes with tandem repeats.The analysis revealed that 38 genes (55.7%) formed 15 tandem repeat clusters.Among these clusters, the A09 chromosomes exhibited the highest number of tandem repeat clusters, with a total of four clusters.Additionally, the A02, A03, A06, and A07 chromosomes each contained two tandem repeat clusters.On the other hand, the A04, A05, and A10 chromosomes harbored only one tandem repeat cluster each, while no tandem repeats were observed on the A01 and A08 chromosomes.This implies that tandem repeats have a significant impact on the proliferation of the BraGSTs gene family.

Conservative Structure and Gene Structure Analysis of BraGSTs Gene Family
The conserved motifs in B. rapa were analyzed using the Meme online tool (MEME-Submission form (meme-suite.org) accessed on 12 January 2023) (Figure 3B).The analysis predicted a maximum of 10 conserved motifs, with all other settings set to default values.A total of 10 conserved motifs were identified in B. rapa.The BraGSTs gene family was also identified, and the amino acid length of each motif ranged from 15 to 64 amino acids (Table 2).Among these motifs, Motif 5 was found to be highly conserved and present in almost all members of the BraGSTs gene family.This result suggests that Motif 5 plays an essential role and has important functions in the BraGSTs gene family.

Conservative Structure and Gene Structure Analysis of BraGSTs Gene Family
The conserved motifs in B. rapa were analyzed using the Meme online tool (MEME-Submission form (meme-suite.org) accessed on 12 January 2023) (Figure 3B).The analysis predicted a maximum of 10 conserved motifs, with all other settings set to default values.A total of 10 conserved motifs were identified in B. rapa.The BraGSTs gene family was also identified, and the amino acid length of each motif ranged from 15 to 64 amino acids (Table 2).Among these motifs, Motif 5 was found to be highly conserved and present in almost all members of the BraGSTs gene family.This result suggests that Motif 5 plays an essential role and has important functions in the BraGSTs gene family.The gene structure maps of BraGSTs were generated using TBtools software(TBtools v1.132).BraGSTU8 lacked a UTR, while BraGSTF11 contained one UTR.All members of the BraGSTs gene family possessed a coding sequence (CDS).The quantity of CDS varied significantly among different BraGSTs genes.Specifically, BraGSTU19 had one CDS, BraGSTL1 within the Lambda subfamily had 15 CDS, and BraGSTL2 had 8 CDS, making it the subfamily with the highest intron distribution.These results indicate that the DHAR and Phi subfamilies in B. rapa exhibit greater conservation.Genes 2023, 14, 1689 6 of 16

Analysis of cis-Acting Elements in BraGSTs Gene Family
A total of 63 cis-acting elements were identified within the promoter region of the BraGSTs gene family.These elements were categorized into nine functional groups, with light response elements comprising the majority, followed by promoter and enhancer elements, low-temperature stress response elements, and plant hormone response elements (Figure 4).Notably, CAAT-box and TATA-box were identified as the most significant elements within promoters and enhancers, while LTR and ARE constituted the largest proportion of cryogenic response components.The ABRE and CGTCA-motif response elements were the most prevalent in plant hormone responses.The presence of MYB, MYC, and MYB-like response elements was abundant in both biotic and abiotic stress responses.Certain genes exhibit endosperm-specific expression elements, namely GCN4-motif and AACA-motif.Additionally, there were genes with meristem-specific expression elements, such as CATbox and RY-element.Some genes possess protein binding sites, including AT-rich elements and BOX III.The response elements associated with low-temperature stress encompass ARE, GC-motif, GTGGC-motif, LTR, MBS, TC-rich, W box, and WUN-motif.
STL1 within the Lambda subfamily had 15 CDS, and BraGSTL2 had 8 CDS, making it the subfamily with the highest intron distribution.These results indicate that the DHAR and Phi subfamilies in B. rapa exhibit greater conservation.

Analysis of cis-Acting Elements in BraGSTs Gene Family
A total of 63 cis-acting elements were identified within the promoter region of the BraGSTs gene family.These elements were categorized into nine functional groups, with light response elements comprising the majority, followed by promoter and enhancer elements, low-temperature stress response elements, and plant hormone response elements (Figure 4).Notably, CAAT-box and TATA-box were identified as the most significant elements within promoters and enhancers, while LTR and ARE constituted the largest proportion of cryogenic response components.The ABRE and CGTCA-motif response elements were the most prevalent in plant hormone responses.The presence of MYB, MYC, and MYB-like response elements was abundant in both biotic and abiotic stress responses.Certain genes exhibit endosperm-specific expression elements, namely GCN4-motif and AACA-motif.Additionally, there were genes with meristem-specific expression elements, such as CAT-box and RY-element.Some genes possess protein binding sites, including AT-rich elements and BOX III.The response elements associated with low-temperature stress encompass ARE, GC-motif, GTGGC-motif, LTR, MBS, TC-rich, W box, and WUNmotif.

Subcellular Localization of BraGSTF2 Protein
The tissue and cell sites where the BraGSTF2 protein was predicted using an online website (https://wolfport.hgc.jp/,accessed on 12 January 2023).The prediction results indicate that the BraGSTF2 protein is localized in the cytoplasm and cell membrane.To further investigate its subcellular localization, the 35S-BraGSTF2-GFP subcellular localization vector was introduced into tobacco plants, while the 35S-GFP empty vector served as the control.The plants were incubated overnight for documentation purposes.After 2-3 days, the epidermis was dissected, and the fluorescence was visualized using a laser confocal microscope.The obtained results are presented in Figure 6.Furthermore, alongside the conspicuous green fluorescence observed in the cell membrane, the BraGSTF2 protein exhibits evident fluorescence within the nucleus, while no fluorescence is detected in other cellular compartments.These findings indicate that the expression of the BraGSTF2 protein in tobacco leaves is likely localized within the cell membrane and nucleus, aligning with the anticipated outcomes.However, additional validation of alternative techniques is warranted in subsequent investigations.The color scale represents the relative expression levels from low (blue) to high (red).

Subcellular Localization of BraGSTF2 Protein
The tissue and cell sites where the BraGSTF2 protein was predicted using an online website (https://wolfport.hgc.jp/,accessed on 12 January 2023).The prediction results indicate that the BraGSTF2 protein is localized in the cytoplasm and cell membrane.To further investigate its subcellular localization, the 35S-BraGSTF2-GFP subcellular localization vector was introduced into tobacco plants, while the 35S-GFP empty vector served as the control.The plants were incubated overnight for documentation purposes.After 2-3 days, the epidermis was dissected, and the fluorescence was visualized using a laser confocal microscope.The obtained results are presented in Figure 6.Furthermore, alongside the conspicuous green fluorescence observed in the cell membrane, the BraGSTF2 protein exhibits evident fluorescence within the nucleus, while no fluorescence is detected in other cellular compartments.These findings indicate that the expression of the BraGSTF2 protein in tobacco leaves is likely localized within the cell membrane and nucleus, aligning with the anticipated outcomes.However, additional validation of alternative techniques is warranted in subsequent investigations.

Screening and Identification of Transgenic Arabidopsis in BraGSTF2 Gene
Following the infection of Arabidopsis, the plants were subjected to antibiotic treatment for three successive generations.The T3 generations of transgenic Arabidopsis plants were subsequently transplanted into flowerpots, where they exhibited normal growth for the subsequent experiment (Supplementary Figure S1A,B).Positive plants with overexpression of BraGSTF2 yielded target bands of approximately 700 bp, whereas wild-type Arabidopsis plants and water samples did not exhibit band amplification.This observation confirmed the successful transfer of the BraGSTF2 target gene into Arabidopsis, resulting in the generation of a positive transgenic plant (Supplementary Figure S2).
The RNA of four transgenic single plants overexpressing BraGSTF2 was extracted and quantitatively analyzed using fluorescence.The results demonstrated that the relative expression of the BraGSTF2 gene in the overexpressed plants was significantly higher compared to the WT.Notably, the G1 single plant exhibited the highest relative expression, which was 22.9 times that of the wild type.Conversely, the G3 single plant displayed the lowest relative gene expression, 6.8 times that of the wild type.The relative gene expression of GSTF2 in the G3 single plant was found to be the lowest, measuring 6.8 times that of the WT.This observation, along with the fact that the other three single plants exhibited a relative gene expression of GSTF2 22.9 times that of the WT, suggests that all four single plants can be classified as BraGSTF2 overexpressed plants (Supplementary Figure S3).

Phenotypic and Survival Analysis of WT and BraGSTF2 Transgenic Arabidopsis under Low-Temperature Stress
WT and transgenic plants were subjected to a −4 °C treatment for varying durations of 3 h, 6 h, 12 h, and 24 h, followed by normal growth conditions for 7 days.The results demonstrate that both WT and transgenic plants exhibited normal growth after being exposed to low temperatures for 3 h and 6 h.However, the WT plants subjected to a 12 h treatment experienced significant mortality, whereas the majority of the transgenic plants managed to survive under the same low-temperature conditions.Furthermore, a small proportion of transgenic plants survived even after 24 h exposure to low temperatures, as illustrated in Figure 7.The results of the statistical analysis conducted on the survival rate

Screening and Identification of Transgenic Arabidopsis in BraGSTF2 Gene
Following the infection of Arabidopsis, the plants were subjected to antibiotic treatment for three successive generations.The T3 generations of transgenic Arabidopsis plants were subsequently transplanted into flowerpots, where they exhibited normal growth for the subsequent experiment (Supplementary Figure S1A,B).Positive plants with overexpression of BraGSTF2 yielded target bands of approximately 700 bp, whereas wild-type Arabidopsis plants and water samples did not exhibit band amplification.This observation confirmed the successful transfer of the BraGSTF2 target gene into Arabidopsis, resulting in the generation of a positive transgenic plant (Supplementary Figure S2).
The RNA of four transgenic single plants overexpressing BraGSTF2 was extracted and quantitatively analyzed using fluorescence.The results demonstrated that the relative expression of the BraGSTF2 gene in the overexpressed plants was significantly higher compared to the WT.Notably, the G1 single plant exhibited the highest relative expression, which was 22.9 times that of the wild type.Conversely, the G3 single plant displayed the lowest relative gene expression, 6.8 times that of the wild type.The relative gene expression of GSTF2 in the G3 single plant was found to be the lowest, measuring 6.8 times that of the WT.This observation, along with the fact that the other three single plants exhibited a relative gene expression of GSTF2 22.9 times that of the WT, suggests that all four single plants can be classified as BraGSTF2 overexpressed plants (Supplementary Figure S3).

Phenotypic and Survival Analysis of WT and BraGSTF2 Transgenic Arabidopsis under Low-Temperature Stress
WT and transgenic plants were subjected to a −4 • C treatment for varying durations of 3 h, 6 h, 12 h, and 24 h, followed by normal growth conditions for 7 days.The results demonstrate that both WT and transgenic plants exhibited normal growth after being exposed to low temperatures for 3 h and 6 h.However, the WT plants subjected to a 12 h treatment experienced significant mortality, whereas the majority of the transgenic plants managed to survive under the same low-temperature conditions.Furthermore, a small proportion of transgenic plants survived even after 24 h exposure to low temperatures, as illustrated in Figure 7.The results of the statistical analysis conducted on the survival rate of seedlings subjected to low-temperature stress indicate that both WT and transgenic plants treated at −4 • C for 3 h and 6 h exhibited normal growth.However, a significant disparity in survival rates was observed between WT and transgenic plants after 12 h of treatment.Specifically, the survival rate of WT plants was recorded at 2.1%, whereas the survival rate of transgenic plant seedlings reached 75.1% (Figure 8).Furthermore, all WT plants perished after 24 h, while the survival rate of transgenic plants was measured at 43.3%.
Genes 2023, 14, x FOR PEER REVIEW 9 of 16 of seedlings subjected to low-temperature stress indicate that both WT and transgenic plants treated at −4 °C for 3 h and 6 h exhibited normal growth.However, a significant disparity in survival rates was observed between WT and transgenic plants after 12 h of treatment.Specifically, the survival rate of WT plants was recorded at 2.1%, whereas the survival rate of transgenic plant seedlings reached 75.1% (Figure 8).Furthermore, all WT plants perished after 24 h, while the survival rate of transgenic plants was measured at 43.3%.Genes 2023, 14, x FOR PEER REVIEW 9 of 16 of seedlings subjected to low-temperature stress indicate that both WT and transgenic plants treated at −4 °C for 3 h and 6 h exhibited normal growth.However, a significant disparity in survival rates was observed between WT and transgenic plants after 12 h of treatment.Specifically, the survival rate of WT plants was recorded at 2.1%, whereas the survival rate of transgenic plant seedlings reached 75.1% (Figure 8).Furthermore, all WT plants perished after 24 h, while the survival rate of transgenic plants was measured at 43.3%.

Physiological and Biochemical Index Analysis of Arabidopsis under Low-Temperature Stress
In this study, Arabidopsis leaves were utilized to assess physiological and biochemical indicators to evaluate the impact of low-temperature treatment at −4 • C on plant damage.The findings revealed that the activities of CAT and SOD, as well as the SP content, initially increased and subsequently decreased in both wild-type (WT) and transgenic plants.Conversely, the MDA content consistently increased throughout the low-temperature stress.At 25 • C, the CAT activity, SOD activity, and MDA content of the WT plants exhibited minimal changes in comparison to the transgenic plants.However, the SP content of the WT plants was higher than that of the transgenic plants.
After a 3 h low-temperature treatment, the catalase (CAT) activity, superoxide dismutase (SOD) activity, and soluble protein (SP) content of the wild type (WT) were found to be higher than those of the transgenic plant (G), exhibiting a 52.6% increase compared to the initial measurement.Conversely, the malondialdehyde (MDA) content of the WT was significantly higher, with a 51.5% difference compared to the transgenic plant (G).However, the CAT activity, SOD activity, and SP content did not show a significant increase after 6 h of low-temperature treatment compared to the initial measurement.Following a 6-h low-temperature treatment, the WT exhibited a 12.2% increase in CAT activity, a 16.7% increase in SOD activity, and an increase in SP content compared to the transgenic plant (G).
Following a 12 h treatment at −4 • C, the activities of CAT, POD, and the content of SP reached their peak values, exhibiting a respective increase of 16.1%, 32.8%, and 11.4% compared to the WT.However, as the processing time prolonged, the activities of CAT, SOD, and the content of SP declined, while the content of MDA continued to rise (Figure 9).

Physiological and Biochemical Index Analysis of Arabidopsis under Low-Temperature Stress
In this study, Arabidopsis leaves were utilized to assess physiological and biochemical indicators to evaluate the impact of low-temperature treatment at −4 °C on plant damage.The findings revealed that the activities of CAT and SOD, as well as the SP content, initially increased and subsequently decreased in both wild-type (WT) and transgenic plants.Conversely, the MDA content consistently increased throughout the low-temperature stress.At 25 °C, the CAT activity, SOD activity, and MDA content of the WT plants exhibited minimal changes in comparison to the transgenic plants.However, the SP content of the WT plants was higher than that of the transgenic plants.
After a 3 h low-temperature treatment, the catalase (CAT) activity, superoxide dismutase (SOD) activity, and soluble protein (SP) content of the wild type (WT) were found to be higher than those of the transgenic plant (G), exhibiting a 52.6% increase compared to the initial measurement.Conversely, the malondialdehyde (MDA) content of the WT was significantly higher, with a 51.5% difference compared to the transgenic plant (G).However, the CAT activity, SOD activity, and SP content did not show a significant increase after 6 h of low-temperature treatment compared to the initial measurement.Following a 6-h low-temperature treatment, the WT exhibited a 12.2% increase in CAT activity, a 16.7% increase in SOD activity, and an increase in SP content compared to the transgenic plant (G).
Following a 12 h treatment at −4 °C, the activities of CAT, POD, and the content of SP reached their peak values, exhibiting a respective increase of 16.1%, 32.8%, and 11.4% compared to the WT.However, as the processing time prolonged, the activities of CAT, SOD, and the content of SP declined, while the content of MDA continued to rise (Figure 9).

Discussion
Glutathione S-transferase plays an important role in plant growth and development and stress management [27].Genome-wide analysis indicated that 70 GSTs genes from B. rapa were divided into eight subclasses based on the domain information and phylogenetic analysis.The Tau subclass was numerous, with 37 genes and Phi with 22 (Figure 1 and Table 1).All genes are unevenly distributed on ten chromosomes (Figure 2).Most genes in the same evolutionary branch contained the same conserved motif, which was similar to that of B. rapa (Figure 3B).

Discussion
Glutathione S-transferase plays an important role in plant growth and development and stress management [27].Genome-wide analysis indicated that 70 GSTs genes from B. rapa were divided into eight subclasses based on the domain information and phylogenetic analysis.The Tau subclass was numerous, with 37 genes and Phi with 22 (Figure 1 and Table 1).All genes are unevenly distributed on ten chromosomes (Figure 2).Most genes in the same evolutionary branch contained the same conserved motif, which was similar to that of B. rapa (Figure 3B).
The subclass of exon and intron in the GSTs gene family was determined through cluster analysis and gene structure analysis (Figure 3C).Previous studies have demonstrated the significant involvement of these two types of GSTs genes in plant resistance to abiotic stress [28].Furthermore, a hormone stress-specific cis-regulatory element was identified in the promoter region of the GSTs gene, suggesting its involvement in hormone regula-tion, as illustrated in Figure 4.These findings establish a theoretical foundation for future investigations into the functionality of the GSTs gene family, particularly in eukaryotes.
To comprehend the reaction of GSTs genes to low-temperature stress, the expression levels of 15 genes were observed to be significantly altered, either upregulated or downregulated (Figure 5).Cold-tolerant varieties exhibited elevated GST enzyme activity and expression levels of 28 stress-related genes when subjected to low-temperature stress.Certain CmGSTs, specifically those classified as Tau, Phi, and DHAR, were found to play a crucial role in response to cold stress.The study suggests that the JrGSTU1 transgenic walnut has the potential to enhance its cold tolerance by upregulating the expression of the GSTs gene and increasing GST enzyme activity under cold stress conditions [29].Additionally, Ma et al. [4] observed a significant upregulation of BnaA02g35760D, BnaC06g20450D, BnaC06g35490D, BnaA02g03230D, and BnaA02g35980D genes in strong cold-resistant walnut varieties, with expression levels 7-12 times higher compared to weak cold-resistant varieties under cold stress.The study found that Arabidopsis plants overexpressing GSTF2 under low-temperature stress were stronger than WT plants, and overexpressing GSTF2 in Korean rape enhanced the tolerance of plants [30].Harshavardhanan et al. found that under low-temperature stress, the expression of BoGSTF10 and BoGSTU24 genes in cabbage was up-regulated [19].In the third chapter, the proteomic study found that the protein expression abundance of the GSTF2 gene in Longyou-7 was 1.6.The GSTF2 gene also participated in the response to low-temperature stress, but its functional mechanism needs further study.
The subcellular localization findings indicated that GSTF2 exhibited presence in both the cell membrane and the nucleus, as depicted in Figure 6.Consequently, further investigation is required to elucidate its specific functionalities.To this end, the GSTF2 gene was introduced into Arabidopsis, resulting in overexpression.Subsequent exposure to low-temperature stress revealed that the transgenic plants exhibited a higher survival rate in comparison to the wild type, as illustrated in Figures 7 and 8. Subsequent analysis of physiological and biochemical parameters in the transgenic plants subjected to low-temperature stress demonstrated a significant increase in enzyme activity and soluble protein content relative to the overexpressed plants (Figure 9).Previous research conducted on Arabidopsis has demonstrated that AtGSTF2 lacks enzymatic catalytic activity but functions as a ligand-protein by directly binding with auxin [31].This interaction plays a crucial role in regulating the distribution and accumulation of auxin within plants.Furthermore, this finding provides evidence for the association between auxin and the differentiation of stem tip meristem [32].Additionally, it was observed that these plants exhibit remarkable tolerance to freezing conditions, enabling them to adapt effectively to such harsh environmental circumstances.
The findings of this study demonstrate that the expression of the GSTF2 gene in transgenic plants was significantly elevated compared to the wild type under low-temperature stress [33].This study further supports the notion that the GSTF2 gene plays a crucial role in enhancing the cold resistance of plants.Consequently, these results offer valuable insights into the underlying mechanisms of cold resistance in winter B. rapa and provide a foundation for enhancing the cold resistance of northern winter rapeseed.

Identification of Members of BraGSTs Gene Family in B. rapa
The B. rapa genome and gene structure annotation information, exhibiting significant homology, were acquired from the B. rapa database (http://brassicadb.cn/,accessed on 12 January 2023) [34].The BraGSTs domain (PF00043 and PF02798) was obtained from the Pfam database (https://www.ebi.ac.uk/interpro/, accessed on 12 January 2023) [24].The HMMER software was utilized to search the Longyou-7 database, resulting in the retrieval of protein sequences containing the PF00043 and PF02798 domains [34].At the same time, the online software ExPASy (http://web.expasy.org/protparam/,accessed on 12 January 2023) was used to predict the amino acid number, molecular weight, isoelectric point, hydrophilicity, and fat index of all family members [35,36].ClustaW was used for multi-sequence alignment of BraGSTs amino acid sequences.Based on multiple sequence alignment, the cluster diagram is constructed by the adjacency method (Neighbor-Join, NJ) in MEGA7.0 software [37].
The initial coordinates of each BraGSTs gene on the chromosome, along with other pertinent details, were acquired from the B. rapa genome database.The physical positioning of these genes on the chromosome was then mapped using MapChart software [38].The BraGSTs gene sequences were obtained, and the promoter sequences of candidate BraGSTs genes in B. rapa, spanning 2000 bp upstream, were extracted.The cis-acting elements were subsequently analyzed using the PlantCARE database (http://bioinformatics.psb.ugent.be/webtools/plantcare/html/, accessed on 12 January 2023), and the corresponding charts were generated [39].The conserved motif of the BraGSTs protein in B. rapa was examined using the online analysis tool MEME (http://meme-suite.org,accessed on 12 January 2023).A maximum of 10 motifs were considered, while the default parameters were utilized for all other settings [40].Fragment replication and tandem replication of the B. rapa GSTs gene family were analyzed using MCScanX software (made with default parameter values) [41].

Prediction of BraGSTs Expression under Low-Temperature Stress
The expression of the BraGSTs gene family was predicted and analyzed using the transcriptome data of "Longyou-7" provided by the rape laboratory of Gansu Agricultural University.Additionally, the heat map was generated using TBtools software [42].The time points S1 and S4 corresponded to the overwintering period on 13 October (0 • C) and 16 December (−11 • C), respectively.

Preliminary Verification of BraGSTF2 Gene Function
The BraGSTF2 gene was cloned by PCR based on specific primers (BraGSTF2-F: ATG-GCAGGTATCAAAGTTTTCG; BraGSTF2-R: CTGAAGGATCTTCTGTGAAGC). Concerning Homologous Recombination technology, PCR amplification was carried out using primers (BraGSTF2-F: CGGGGGACGAGCTCGGTACCATGGCAGGTATCAAA GTTTTCG; BraGSTF2-R: ACCATGGTGTCGACTCTAGACTGAAGGAT CTTCTGTGAA GC).The recovered products were ligated with BP Clonase enzyme (Invitrogen, Carlsbad, CA, USA) and the pDONR vector.After transforming into coliform bacteria (DH5α), the positive clones were screened by smear sequencing.The positive clones were selected to interact with LR Clonase enzyme (Invitrogen, Carlsbad, CA, USA) to connect to the overexpression vector pEarly-Gate101, following the instructions for Agrobacterium tumefaciens GV3101 (Anyu Biotechnology Co., Ltd., Shanghai, China).After the bacteria were picked out from the coated plate, colony PCR was carried out, and the positive bacterial solution was selected and preserved [43].
Transgenic Arabidopsis was obtained by the floral-dip method.The T3 seeds were obtained by screening with antibiotics.The T3 seeds of transgenic Arabidopsis lines and WT Arabidopsis were sown in pots according to the experimental requirements.The phenotypes of WT and transgenic Arabidopsis plants treated at −4 • C were observed at the seedling stage.WT and transgenic plants were grown for about 30 days and were treated in a −4 • C incubator for 3 h, 6 h, 12 h, and 24 h, respectively.Four plants were taken in each pot, and the other part resumed normal growth, and the plant phenotype and survival rate were observed and counted after one week.The leaves of the plants were frozen in liquid nitrogen and stored in the cryogenic refrigerator.The CAT activity and SOD activity, MDA content, and SP content were determined, and the rest of Arabidopsis recovered.One week later, the plant survival rate was observed and counted [44,45].The CAT activity and SOD activity, MDA content, and SP content were estimated according to the method developed by Gill et al. [46] and Quiroga et al. [47].Three biological replicates were set up for each of the above experimental treatments.

RNA Isolation, Reverse Transcription, qRT-PCR, and T Transcriptome Expression Analysis
RNA Extraction Kits (TIANGEN Biotech Co., Ltd., DP432, Beijing, China) were employed to extract total RNA from both wild-type (WT) and transgenic plants, ensuring the elimination of genomic DNA contamination.The cDNA synthesis was carried out using the PrimeScript™ RT Master Mix (Vazyme, Nanjing, China) following the provided instructions.Subsequently, the quality and concentration of the obtained cDNA were assessed using an ultra-trace UV-visible spectrophotometer (NanoVueTM Plus, Wilmington, DE, USA) and stored in a standby state.
Genes within the transcriptome data were chosen, and specific primers were designed for qRT-PCR based on the guidelines provided by the ChamQ Universal SYBR qPCR Master Mix (Shanghai, China).Primers (BraGSTF2-pcr-F: GCGAACTGTCTGATGCTC and BraGSTF2-pcr-R: CGTATCGGTGAGCTATGTACTG) were used to perform qRT-PCR on Arabidopsis.AtActin2 (F: TGTGCCAATCTACGAGGGTTT; R: TTTCCCGCTCTGCTGTTGT) was used as an internal control for Arabidopsis.The reaction procedure consisted of an initial denaturation step at 95 • C for 10 min, followed by 40 cycles of denaturation at 95 • C for 30 s and annealing/extension at 60 • C for 1 min.Subsequently, a melting curve analysis was conducted within the temperature range of 65-95 • C. The qRT-PCR efficiency of the genes was determined by evaluating the standard curve derived from the gradient dilution of cDNA.To normalize the quantity of template cDNA, the gene fragment encoding Arabidopsis actin RNA was utilized as an internal control.The relative expression values of each gene were calculated using the comparative 2 −∆∆CT method [48].The RNA-seq libraries (SRP179662) and (SRP211768) were selected for gene-expression analysis [44,45].Three biological replicates were set up for each experimental treatment.

Subcellular Localization of BraGSTF2 Protein in Tobacco
Agrobacterium tumefaciens strains containing the PCAMBIA2300-BraGSTF2-GFP construct, and the pCAM-BIA2300-GFP empty vector were transiently introduced into Benji tobacco leaves.The tobacco plants were cultured at 23 • C under dark conditions for 24 h.Subsequently, small sections of tobacco leaves were excised to create thin slices for analysis.The fluorescence patterns of the pCAMBIA2300-BraGSTF2-GFP and pCAM-BIA2300-GFP constructs were visualized using a laser scanning confocal microscope LSCM800 (Carl Zeiss, Jena, Germany) with an excitation wavelength of 488 nm [49].

Conclusions
In this study, a total of 70 members of the BraGSTs gene family were identified and classified into 8 subfamilies.It was observed that members within the same subfamily exhibited identical gene structures and functional motifs.Furthermore, the distribution of these genes across 10 chromosomes was found to be uneven.Based on these findings, it is hypothesized that the BraGSTF2 gene plays a significant role in mitigating the effects of low-temperature stress.Additionally, the subcellular localization of the BraGSTF2 protein was determined to be in the nucleus and cell membrane.Further investigation revealed that the over-Arabidopsis BraGSTF2 gene resulted in higher levels of CAT, SOD activity, and SP content compared to the wild type, while the MDA content was lower under lowtemperature stress conditions.These results suggest that the BraGSTF2 gene may have a regulatory function in response to low-temperature stress.

Figure 1 .
Figure 1.Clustering analysis of BraGSTs gene family in B. rapa.Eight subfamilies and branches are marked with different colors.

Figure 1 .
Figure 1.Clustering analysis of BraGSTs gene family in B. rapa.Eight subfamilies and branches are marked with different colors.

Figure 2 .
Figure 2. Chromosomal location and gene duplication in B. rapa.The tandem duplicated genes are marked with hot pink.The chromosome number is indicated at the top of each chromosome; Chr00 is the gene scaffold.The scales on the left and right sides (in Mb) are for the chromosomes and scaffold, respectively.

Figure 2 .
Figure 2. Chromosomal location and gene duplication in B. rapa.The tandem duplicated genes are marked with hot pink.The chromosome number is indicated at the top of each chromosome; Chr00 is the gene scaffold.The scales on the left and right sides (in Mb) are for the chromosomes and scaffold, respectively.

Figure 3 .
Figure 3. Clustering analysis, conserved structure, and gene structure analysis of the BraGSTs gene in B. rapa.(A) Using MEGA7 to draw a clustering analysis using the maximum likelihood method with default parameters in BraGSTs proteins.(B) Distribution of conserved motifs in the BraGSTs proteins.Boxes of different colors represent 10 putative motifs.(C) Exon/intron organization of the BraGSTs genes.The yellow box indicates the CDS, the green box indicates the UTR, and the black line indicates the intron-scale bars represented as bp.The gene structure maps of BraGSTs were generated using TBtools software(TBtools v1.132).BraGSTU8 lacked a UTR, while BraGSTF11 contained one UTR.All members of the BraGSTs gene family possessed a coding sequence (CDS).The quantity of CDS varied

Figure 3 .
Figure 3. Clustering analysis, conserved structure, and gene structure analysis of the BraGSTs gene in B. rapa.(A) Using MEGA7 to draw a clustering analysis using the maximum likelihood method with default parameters in BraGSTs proteins.(B) Distribution of conserved motifs in the BraGSTs proteins.Boxes of different colors represent 10 putative motifs.(C) Exon/intron organization of the BraGSTs genes.The yellow box indicates the CDS, the green box indicates the UTR, and the black line indicates the intron-scale bars represented as bp.

Figure 4 .
Figure 4. Frequency of cis-regulatory elements in the upstream regions of BraGSTs protein in B. rapa.Scale bars represent bp.

Figure 4 .
Figure 4. Frequency of cis-regulatory elements in the upstream regions of BraGSTs protein in B. rapa.Scale bars represent bp.

Figure 5 .
Figure 5.The heat map of BraGSTs genes expression profile under freezing stress in B. rapa.The values of RNA-seq were transformed using log2, and the heat map was generated by TBtools software.The S1 and S4 represented the overwintering period on 13 October (0 °C) and 16 December (−11 °C).The color scale represents the relative expression levels from low (blue) to high (red).

Figure 5 .
Figure 5.The heat map of BraGSTs genes expression profile under freezing stress in B. rapa.The values of RNA-seq were transformed using log2, and the heat map was generated by TBtools software.The S1 and S4 represented the overwintering period on 13 October (0 • C) and 16 December (−11 • C).The color scale represents the relative expression levels from low (blue) to high (red).

Figure 6 .
Figure 6.The figures presented above portray the subcellular localization of the BraGSTF2 protein within tobacco cells.Progressing from left to right, these figures illustrate the observations of subcellular localization for the 35S-GFP empty vector in distinct fields: dark field, bright field, and the merged field, respectively.Similarly, the figures below showcase the subcellular localization observations of the 35S-BraGSTF2-GFP recombinant vector in three distinct fields, also arranged from left to right.For reference, the scale bars indicate a length of 20 µm, facilitating size assessment.In this context, GFP corresponds to the green fluorescent protein, DIC pertains to bright field microscopy, and Merged signifies an overlaid photograph combining two channels to provide a comprehensive visual representation.

Figure 6 .
Figure 6.The figures presented above portray the subcellular localization of the BraGSTF2 protein within tobacco cells.Progressing from left to right, these figures illustrate the observations of subcellular localization for the 35S-GFP empty vector in distinct fields: dark field, bright field, and the merged field, respectively.Similarly, the figures below showcase the subcellular localization observations of the 35S-BraGSTF2-GFP recombinant vector in three distinct fields, also arranged from left to right.For reference, the scale bars indicate a length of 20 µm, facilitating size assessment.In this context, GFP corresponds to the green fluorescent protein, DIC pertains to bright field microscopy, and Merged signifies an overlaid photograph combining two channels to provide a comprehensive visual representation.

Figure 7 .
Figure 7.The phenotype of WT and BraGSTF2 transgenic Arabidopsis after low-temperature stress.WT: wild type, G: BraGSTF2 transgenic Arabidopsis.

Figure 8 .
Figure 8.The survival rate of plants after low-temperature stress.WT: wild type, G: BraGSTF2 transgenic Arabidopsis; Different lowercase letters represent significant differences in low-temperature treatment times.* indicated that there were significant differences among varieties (p < 0.05), and ** indicated that there were extremely significant differences among varieties (p < 0.01).The error bar represents the standard error of the average of the sample.

Figure 7 .
Figure 7.The phenotype of WT and BraGSTF2 transgenic Arabidopsis after low-temperature stress.WT: wild type, G: BraGSTF2 transgenic Arabidopsis.

Figure 7 .
Figure 7.The phenotype of WT and BraGSTF2 transgenic Arabidopsis after low-temperature stress.WT: wild type, G: BraGSTF2 transgenic Arabidopsis.

Figure 8 .
Figure 8.The survival rate of plants after low-temperature stress.WT: wild type, G: BraGSTF2 transgenic Arabidopsis; Different lowercase letters represent significant differences in low-temperature treatment times.* indicated that there were significant differences among varieties (p < 0.05), and ** indicated that there were extremely significant differences among varieties (p < 0.01).The error bar represents the standard error of the average of the sample.

Figure 8 .
Figure 8.The survival rate of plants after low-temperature stress.WT: wild type, G: BraGSTF2 transgenic Arabidopsis; Different lowercase letters represent significant differences in low-temperature treatment times.* indicated that there were significant differences among varieties (p < 0.05), and ** indicated that there were extremely significant differences among varieties (p < 0.01).The error bar represents the standard error of the average of the sample.

Figure 9 .
Figure 9.Effect of low-temperature stress on physiological indexes in transgenic and WT plants.(A) CAT activities; (B) SOD activities; (C) MDA content; (D) SP content.WT: wild type, G: BraGSTF2 transgenic Arabidopsis; Different lowercase letters represent significant differences in low-temperature treatment times.* indicated that there were significant differences among varieties (p < 0.05).The error bar represents the standard error of the average of the sample.

Figure 9 .
Figure 9.Effect of low-temperature stress on physiological indexes in transgenic and WT plants.(A) CAT activities; (B) SOD activities; (C) MDA content; (D) SP content.WT: wild type, G: BraGSTF2 transgenic Arabidopsis; Different lowercase letters represent significant differences in low-temperature treatment times.* indicated that there were significant differences among varieties (p < 0.05).The error bar represents the standard error of the average of the sample.

Table 1 .
The BraGSTs gene family was classified based on their domain and phylogenetics in B. rapa.

Table 1 .
The BraGSTs gene family was classified based on their domain and phylogenetics in B. rapa.

Table 2 .
Amino acid sequence and amino acid number of Motif.

Table 2 .
Amino acid sequence and amino acid number of Motif.