The Glutathione Peroxidase Gene Family in Nitraria sibirica: Genome-Wide Identification, Classification, and Gene Expression Analysis under Stress Conditions

Plant glutathione peroxidases (GPXs) are the main enzymes in the antioxidant defense system that sustain H2O2 homeostasis and normalize plant reaction to abiotic stress conditions. However, the genome-wide identification of the GPX gene family and its responses to environmental stresses, especially salt stress, in Nitraria sibirica, which is a shrub that can survive in saline environments, has not yet been reported. Here, we first report the genome-wide analysis of the GPX gene family in N. sibirica, leading to a total of seven NsGPX genes that are distributed on six of the twelve chromosomes. Phylogenetic analysis showed that NsGPX genes were grouped into four major groups (Group I-IV). Three types of cis-acting elements were identified in the NsGPX promoters, mainly related to hormones and stress response. The quantitative real-time PCR (qRT-PCR) analysis indicated that NsGPX1 and NsGPX3 were significantly up-regulated in stem and leaf, while NsGPX7 transcriptionally in root in response to salt stress. The current study identified a total seven NsGPX genes in N. sibirica via genome-wide analysis, and discovered that NsGPXs may play an important role in response to salt stress. Taken together, our findings provide a basis for further functional studies of NsGPX genes, especially in regarding to the resistance to salt stress of this halophyte plant N. sibirica, eventually aid in the discovery of new methods to restore overtly saline soil.


Introduction
Reactive oxygen species (ROS), known as free radicals like O 2− , OH and non-radicals like H 2 O 2 and 1O 2 , are originated from incomplete reduction of molecular oxygen in aerobic organisms [1]. Endogenously produced ROS under abiotic stress conditions usually serve as cellular messengers and redox regulators involved in a number of biological processes in plants, but significant accumulation of ROS can cause damage to cellular apparatuses and macromolecules, and eventually program the cells' death [2]. In many plants, the apoplast, mitochondria, plasma membrane, chloroplast, peroxisomes, endoplasmic reticulum, and cell walls are the primary sites of ROS production [3]. Intracellular ROS are mainly produced at a low level in plants under optimal growth conditions [4]. Previous research revealed that overproduction of ROS produced by abiotic stress conditions in plant cells is extremely reactive and toxic to proteins, lipids, and nucleic acids, which eventually causes cell destruction and death [5]. However, on the other hand, it is also believed that the increased ROS generation in response to stress might work as a signal to activate plant stress response pathways [6]. Therefore, plant cells have evolved sophisticated antioxidant systems to maintain a dynamic balance between ROS production and elimination, thereby minimizing the ROS damage to cells [7]. ROS-scavenging enzymes of plants mainly including glutathione peroxidase (GPX), ascorbate peroxidase (APX), catalase (CAT), superoxide dismutase (SOD), etc. [8]. Among these different ROS-scavenging enzymes, the term glutathione peroxidase (EC 1.11.1.9 for classical glutathione peroxidase and EC 1.11.1.12, phospholipid-hydroperoxide glutathione peroxidase) was introduced by Mills who discovered the reaction with H 2 O 2 in enzyme preparations from mammalian red blood cells [9]. Compared with mammals GPXs, plant GPXs include cysteine as opposed to selenocysteine in their active site and some of them contain both glutathione peroxidase and thioredoxin peroxidase functions [10]. The first study of plant GPXs is the cloning and characterization of the cDNA from Nicotiana sylvestris, which is highly similar to animal GPXs except lacks selenocysteine encoded by the terminal TGA [11]. Plant GPXs were discovered to form a distinct cluster by means of a worldwide phylogenetic analysis, indicating that a single ancestral gene may have been the source of all plant GPX genes [12].
The GPX gene families have been discovered and analyzed in numerous plants, such as Citrus sinensis [13], Arabidopsis thaliana [14], Lycopersicon esculentum [15] and so on. Several related researches have shown that GPX gene help plant to adaptation to various abiotic stresses in multiple ways. In A. thaliana, eight members of GPX gene family were identified by many researches, four of them were up-regulation under salt stress [16]. Besides, the well-preserved germination rate, seedling growth and chlorophyll content of the AtGPXL5-overexpressing seedlings under the stress of 100 mM NaCl indicating the increased salt tolerance of the plant [17]. The up-regulation of OsGPX2 and OsGPX4 gene helps several plants to response to drought and oxidative stress, while down-regulated under high salt, heat shock and cold stress in rice (Oryza sativa L.) [18]. Under salinity stress, the majority of CsGPX genes were down-regulated at particular time points in cucumber (Cucumis sativus L.) [19]. Six ClGPX genes were identified in watermelon (Citrullus lanatus), all of them were obviously up-regulated under salty stress [20]. Different members of eight TsGPX genes were coordinately regulated under certain environmental stress circumstances, supporting the critical functions of TsGPXs in salt and drought stress response in Thellungiella salsuginea [21]. Research in desert poplar (Populus euphratica Oliv.) suggested that the GPX gene family genes with induced gene expression pattern belong to a single gene family and are abiotic defend responding to salt stress [22]. Exceptional, in some cases, the expression levels of specific GPX genes may decrease in specific stressed plants, for instance, several GPX genes showed reduced expression in response to drought stress in sorghum (Sorghum bicolor L.), which implies that GPX genes may be involved in the activation of multiple downstream pathways [23]. In summary, these investigations concluded that plant GPXs genes might regulate plant developmental processes, stress reactions, tolerance mechanisms and more [24].
N. sibirica is a typical salt-tolerant shrub that thrives in the desert, saline and coastal saline-alkali lands [25], which is a member of the genus Nitraria Linn., and is found in the Sapindales clade [26]. Related studies have found that N. sibirica belongs to an ecologically adaptabe halophyte with high salt tolerance, which is represented by a certain amount of tissue salt tolerance [27]. These result means N. sibirica is a potential pioneer species in high salt environment with strong saline-alkali resistance. In reality, N. sibirica is widely distributed in Mongolia, Central Asia, Siberia and China. Due to its high drought resistance and adaptability to saline-alkaline settings, it is the perfect plant for the treatment of salinization and alkalinity [28]. To our current knowledge, the GPX gene family has not been investigated in N. sibirica, which has influenced the further study on the regulation process of salt stress resistance of N. sibirica and restoration overtly saline soil. Thus, being the first, the current study carried out an analysis to identify GPX gene family within N. sibirica, and revealed the role of NsGPX in salt resistance physiology.

Plant Materials and Abiotic Stress Treatment
The seeds utilized for this investigation collected from the Inner Mongolia municipality, Dengkou County, China. The seeds were wrapped in wet sand, kept at 4 • C for more than 60 days to vernalize. The seeds were sown at 23 • C, and they took almost a month to grow to a height of around 25 cm in a 4:1:1 soil mixture of nutrient-rich soil, vermiculite, and perlite. Healthy living N. sibirica seedlings of similar growth were given 500 mM NaCl treatment at the same stage of development to mimic salt stress. After 0, 24, and 48 h of salt stress, plant tissues were collected and categorized into root, stem, and leaf sections for subsequent experiments. Following that, every single one of the plant tissues were gathered, quickly frozen with liquid nitrogen, and kept at −80 • C immediately to extract RNA [28]. The dates of sample collection were 15 August to 17 August 2022.

Identification of GPX Genes in N. sibirica
The HMM (Hidden Markov Model) file of the GPX domain (Pfam: PF00255) from the Pfam database (http://pfam.xfam.org/, accessed on 10 August 2022), using a local software, HMMER3.1 [29], to make comparison in the unpublished genome database of N. sibirica. As for BLASTP, we used BLASTP software (v2.90) eight A. thaliana GPXs amino acid sequences as an enquiry by e-value set to 1 × 10 −5 . The amino acid sequences of eight AtGPXs were retrieved from the TAIR Arabidopsis genome database (http://www.arabidopsis.org/, accessed on 10 August 2022) [30]. Seven NsGPX genes were recognized by merging the result of these two methods and named after AtGPXs. In order to confirm the correctness, we used SMART (http://smart.embl-heidelberg.de/, accessed on 10 August 2022) and the online CD-search tool (https://www.ncbi.nlm.nih.gov/Structure/bwrpsb/bwrpsb.cgi, accessed on 10 August 2022).

Phylogenetic Tree Analysis of GPX Proteins in N. sibirica
To clarify the evolutionary relationship of the NsGPX gene family, we generated a phylogenetic tree for N. sibirica, A. thaliana, Vitis vinifera and O. sativa protein sequences. ClustalW served as the tool for sequence alignment, pruning of the sequence was accomplished using trimAL [34], iqtree2 (Bootstrap = 1000) was used to construct evolutionary trees, which were beautified using ggtree2 package in R language.

Expression Analysis of GPX Genes in N. sibirica
The Eastep ® Super Total RNA Extraction Kit (Promega, Shanghai, China) was utilized to extract RNA of experimental seedlings; then the RNA was reverse transcribed into single-stranded cDNA using a reverse-transcription kit (Vazyme, Nanjing, China), which containing DNA clearance, after RNA quality control by gel electrophoresis. Using a LightCycler 480II (Roche, Basel, Switzerland), quantitative real-time PCR (qRT-PCR) was performed with the AceQ qPCR SYBR Green Master Mix (Vazyme, Nanjing, China). In order to detect expression of NsGPXs under salt stress, particular qRT-PCR primers were designed in NCBI (https://www.ncbi.nlm.nih.gov/tools/primer-blast/index.cgi?LINK_ LOC=BlastHome, accessed on 1 May 2022) ( Table S2). As for reference genes, we chose NsHis and NsAct, which maintained stable expression under salt stress [35]. All of the primers were verified the specificity and the optimal annealing temperature by gel electrophoresis. All the primers were specific and the optimal annealing temperature was 57 • C ( Figure S1). To further examine the expression responses of the NsGPX gene family under salt stress since N. sibirica is a well-known halophyte shrub, we treated N. sibirica seedling with 500 mM NaCl solution and collected different tissues for expression quantification of random chosen genes, and the data were normalized based against two reference genes. All the primers used were specific ( Figure S2).

Identification and Characterization of NsGPX Genes
By merging the result of HMM search and BLASTP, we identified 7 GPX genes in N. sibirica, and each individual was given a name based on its Arabidopsis homolog. (Table 1). NsGPX proteins ranged from 144 aa (NsGPX6) to 480 aa (NsGPX3, NsGPX4) in length and their molecular weights (MWs) ranged from 16.06 kDa to 54.09 kDa, while their pI ranged from 5.34 to 9.98, with an average of 7.95. The online prediction of subcellular localization showed that two NsGPX proteins, including NsGPX3 and NsGPX4, were located in mitochondrion or plastid, while NsGPX5 was mostly located in plastid. Others are distributed in other part of the cell: cytoplasm (NsGPX7), nucleus (NsGPX6), extracellular (NsGPX2) and endoplasmic reticulum (NsGPX1) ( Table S3).

Phylogenetic Relationships of NsGPX Genes
In order to reveal the evolutionary history of NsGPX genes, the predicted GPX protein sequences from N. sibirica, A. thaliana, V. vinifera and O. sativa were aligned and then were used to reconstruct the phylogenetic tree by the neighbor-joining (NJ) method. The results of the research demonstrated that there were four major combinations among all GPXs, i.e., Group I-IV ( Figure 1).
In general, GPXs clustering into the same group may possess comparable functions. NsGPXs and GPX proteins from other species were spread in each group. Especially, NsGPX1 and NsGPX6 were clustered into Group IV and II, respectively, while NsGPX3/4 and NsGPX2/5/7 were clustered into Group I and III. Furthermore, the expansion of NsGPX3/4 and NsGPX2/5/7 were both lineage-specific, implying a particular function which might be related to biological characteristics of N. sibirica.
By mapping NsGPX genes into chromosomes, we found that these seven NsGPXs were distributed on six of the twelve chromosomes. Most of them corresponded to one chromosome, except for NsGPX3 and NsGPX4 on Chr4. (Figure 2). In general, GPXs clustering into the same group may possess comparable function NsGPXs and GPX proteins from other species were spread in each group. Especiall NsGPX1 and NsGPX6 were clustered into Group Ⅳ and Ⅱ, respectively, while NsGPX3 and NsGPX2/5/7 were clustered into Group Ⅰ and Ⅲ. Furthermore, the expansion NsGPX3/4 and NsGPX2/5/7 were both lineage-specific, implying a particular functio which might be related to biological characteristics of N. sibirica.
By mapping NsGPX genes into chromosomes, we found that these seven NsGPX were distributed on six of the twelve chromosomes. Most of them corresponded to on chromosome, except for NsGPX3 and NsGPX4 on Chr4. (Figure 2).

Collinearity Analysis of NsGPX Genes
Intraspecies collinearity analysis showed that no gene pair existed among these NsGPXs. Interspecific covariance analysis demonstrated substantial orthologs of the GPX genes when it comes to N. sibirica, A. thaliana and V. vinifera (Figure 3). 5 NsGPX genes exhibited syntenic links with 4 AtGPXs and 4 VvGPXs. Predominantly, most homologues of A. thaliana and V. vinifera have colinear affiliations with NsGPXs, which implies that whole-genome duplication (segmental duplication) events participated in a significant role in NsGPX gene family evolution in the N. sibirica genome. Notably, NsGPX3 and NsGPX4 link with one gene both of A. thaliana and V. vinifera, which means these genes share closer relation.

Collinearity Analysis of NsGPX Genes
Intraspecies collinearity analysis showed that no gene pair existed among these Ns-GPXs. Interspecific covariance analysis demonstrated substantial orthologs of the GPX genes when it comes to N. sibirica, A. thaliana and V. vinifera (Figure 3). 5 NsGPX genes exhibited syntenic links with 4 AtGPXs and 4 VvGPXs. Predominantly, most homologues of A. thaliana and V. vinifera have colinear affiliations with NsGPXs, which implies that whole-genome duplication (segmental duplication) events participated in a significant role in NsGPX gene family evolution in the N. sibirica genome. Notably, NsGPX3 and NsGPX4 link with one gene both of A. thaliana and V. vinifera, which means these genes share closer relation. exhibited syntenic links with 4 AtGPXs and 4 VvGPXs. Predominantly, most homologues of A. thaliana and V. vinifera have colinear affiliations with NsGPXs, which implies that whole-genome duplication (segmental duplication) events participated in a significant role in NsGPX gene family evolution in the N. sibirica genome. Notably, NsGPX3 and NsGPX4 link with one gene both of A. thaliana and V. vinifera, which means these genes share closer relation.

Evaluation of Gene Structures and Conserved Motifs of NsGPX Genes
In order to fully comprehend how the genes in the NsGPX family have expanded, the gene structure (exon and intron arrangement) of NsGPXs was scrutinized. The assessment outcome indicated that the number of exon in each gene varied from 4 (NsGPX6) to 12 (NsGPX3/4). No UTR was predicted for NsGPX2, 6 and 7, while the rest NsGPX genes possessed 5ʹ and 3ʹ UTR.
Conserved domain analysis showed that all seven NsGPX genes contained Thiore-doxin_like superfamily (Motif1) and most of them possessed Motif2 and Motif10 except for NsGPX7. The identical motif type and order between NsGPX3 and NsGPX4 implied similar protein functions of this gene pair (Figure 4). Our analysis concluded that, unexpectedly, GPX family members within a group did not share the exact similar genetic architecture and evolutionary relatives.

Evaluation of Gene Structures and Conserved Motifs of NsGPX Genes
In order to fully comprehend how the genes in the NsGPX family have expanded, the gene structure (exon and intron arrangement) of NsGPXs was scrutinized. The assessment outcome indicated that the number of exon in each gene varied from 4 (NsGPX6) to 12 (NsGPX3/4). No UTR was predicted for NsGPX2, 6 and 7, while the rest NsGPX genes possessed 5' and 3' UTR.
Conserved domain analysis showed that all seven NsGPX genes contained Thiore-doxin_like superfamily (Motif1) and most of them possessed Motif2 and Motif10 except for NsGPX7. The identical motif type and order between NsGPX3 and NsGPX4 implied similar protein functions of this gene pair (Figure 4). Our analysis concluded that, unexpectedly, GPX family members within a group did not share the exact similar genetic architecture and evolutionary relatives.

Expression Patterns of NsGPXs in Response to Salt Stress
The prediction of cis-acting elements revealed that three main types of cis-acting elements, i.e., those related to plant hormone signaling, environmental stress responses, and MYB-binding sites, were enriched in the promoter regions of NsGPXs. The evaluation of cis-regulatory elements in the NsGPXs displays that five phytohormone-correlated comprising auxin, abscisic acid (ABA), gibberellin (GA), methyl jasmonate (MeJA), and salicylic acid (SA), responsive elements comprising MYB binding sites, MBS, etc. (Table S4). All the NsGPX genes contain MYB binding sites in their promoter regions, and most of them possessed cis-elements related to more than one phytohormone, indicating that the NsGPX gene family was extensively involved in plant growth and development, as well as in response to environmental stresses like drought and salinity ( Figure 5).

Expression Patterns of NsGPXs in Response to Salt Stress
The prediction of cis-acting elements revealed that three main types of cis-acting elements, i.e., those related to plant hormone signaling, environmental stress responses, and MYB-binding sites, were enriched in the promoter regions of NsGPXs. The evaluation of cis-regulatory elements in the NsGPXs displays that five phytohormone-correlated comprising auxin, abscisic acid (ABA), gibberellin (GA), methyl jasmonate (MeJA), and salicylic acid (SA), responsive elements comprising MYB binding sites, MBS, etc. (Table S4). All the NsGPX genes contain MYB binding sites in their promoter regions, and most of them possessed cis-elements related to more than one phytohormone, indicating that the NsGPX gene family was extensively involved in plant growth and development, as well as in response to environmental stresses like drought and salinity ( Figure 5).

Expression Patterns of NsGPXs in Response to Salt Stress
The prediction of cis-acting elements revealed that three main types of cis-acting elements, i.e., those related to plant hormone signaling, environmental stress responses, and MYB-binding sites, were enriched in the promoter regions of NsGPXs. The evaluation of cis-regulatory elements in the NsGPXs displays that five phytohormone-correlated comprising auxin, abscisic acid (ABA), gibberellin (GA), methyl jasmonate (MeJA), and salicylic acid (SA), responsive elements comprising MYB binding sites, MBS, etc. (Table S4). All the NsGPX genes contain MYB binding sites in their promoter regions, and most of them possessed cis-elements related to more than one phytohormone, indicating that the NsGPX gene family was extensively involved in plant growth and development, as well as in response to environmental stresses like drought and salinity ( Figure 5). qRT-PCR analysis demonstrated that most members of NsGPX gene family transcriptionally responded to salt stress both in root and shoot from 24 h after salt stress until 48 h.
Notably, the specific salt stress responsive NsGPX genes varied between different tissues. Specifically, NsGPX7 was significantly upregulated 24 h after exposed to salt stress and kept stable at 48 h in root (Figure 6a). In shoot, NsGPX1 and NsGPX3, instead of NsGPX7, were transcriptionally up-regulated in response to salt stress (Figure 6b,c). Interestingly, the transcriptional response of NsGPX1 obviously lagged behind NsGPX3 both in stem and leaf. The expression of NsGPX1 was still kept in low level at 24 h but increased a lot at 48 h after exposed to salt stress, while NsGPX3 already transcriptionally up-regulated at 24 h and peaked at 48 h. In particular, the transcriptional response of NsGPX4 was not similar to NsGPX3 under salt stress, which means these two genes have different expression patterns under salt stress. qRT-PCR analysis demonstrated that most members of NsGPX gene family transcriptionally responded to salt stress both in root and shoot from 24 h after salt stress until 48 h.
Notably, the specific salt stress responsive NsGPX genes varied between different tissues. Specifically, NsGPX7 was significantly upregulated 24 h after exposed to salt stress and kept stable at 48 h in root (Figure 6a). In shoot, NsGPX1 and NsGPX3, instead of NsGPX7, were transcriptionally up-regulated in response to salt stress (Figure 6b,c). Interestingly, the transcriptional response of NsGPX1 obviously lagged behind NsGPX3 both in stem and leaf. The expression of NsGPX1 was still kept in low level at 24 h but increased a lot at 48 h after exposed to salt stress, while NsGPX3 already transcriptionally up-regulated at 24 h and peaked at 48 h. In particular, the transcriptional response of NsGPX4 was not similar to NsGPX3 under salt stress, which means these two genes have different expression patterns under salt stress.

Discussion
ROS are commonly produced in plants under stress, which plays a substantial part in plant physiology. In order to prevent excessive ROS from affecting the normal growth of plants, plants have evolved the ability to neutralize excessive ROS, among which GPXs

Discussion
ROS are commonly produced in plants under stress, which plays a substantial part in plant physiology. In order to prevent excessive ROS from affecting the normal growth of plants, plants have evolved the ability to neutralize excessive ROS, among which GPXs are considered to be the greatest possible ROS scavenger [36]. In Ammopiptanthus nanus, research exhibited that GPX family members might participate in the stress response by contributing to reactive oxygen species scavenging [37]. Evidence from diversified studies on GPXs from different plants further demonstrated their crucial physiological and developmental roles. By way of example, AtGPX isoenzymes may function to detoxify H 2 O 2 and organic hydroperoxides using thioredoxin in vivo and may also be involved in regulation of the cellular redox homeostasis by maintaining the thiol/disulfide or NADPH/NADP balance under salt stress [38]. Moreover, every single one with the exception of PpaGPX1 of PpaGPX genes presented up-regulated expression after the highest level of respiratory climacteric in the late stage of ripening, and under heat + 1-MCP treatment, which means PpaGPXs had a more significant regulating function in the later stage of peach fruit ripening [39].
With the goal to understand the response pattern under salt stress of N. sibirica, considered the important role of GPX, genome-wide analyses were performed in N. sibirica to identify GPX gene family members and their expression patterns in response to salt stress. To the best of our current knowledge, the GPX gene family, which responds to salt stress conditions, has not yet been reported in N. sibirica. And we found 7 GPX genes across 6 chromosomes, which can be divided into 4 groups (Figure 1). Previous studies have found that GPX gene family got different pattern of sub-cellular localization [40], while our study showed that NsGPX got similar pattern, just like Populus trichocarpa GPXs [41], this should be further investigated by subcellular localization in the future. Fluctuation in the GPXs gene numbers between distinct species of plants could potentially be attributed to gene replication experiences, study on the Brassica napus GPX gene family revealed that segmental duplication, purifying selection, and positive selection burden did exist during the evolution of BnGPXs [24]. Interspecific covariance analysis uncovered whole-genome duplication events performed a crucial part in NsGPX gene family evolution in the genome. Notably, NsGPX3 and NsGPX4 showed similar location on chromosomal and collinearity, they may be tandem repeat sequences (Figure 2).
The number of motifs varied considerably in the NsGPX gene sequences, ranged from 2 to 11, and the number of CDSs ranged from 5 to 12. Additionally, the discrepancy in the number of introns in the gene structure of GPXs genes implies that some variation exists in this given gene families, which proves that members of the NsGPX gene family are functionally manifold. Remarkably, NsGPX3 and NsGPX4 showed parallel exonintron organization and conserved motifs, which indicates that both of these genes might play a component in the comparable roles pertaining to multiple abiotic cues ( Figure 4). Additionally, similar breakthroughs have been reported on Gossypium hirsutum [42] and Triticum aestivum [43]. Analysis of the cis-acting elements showed that all the NsGPX genes contain MYB binding sites, 4 of NsGPXs contain MYB bonding site associated with drought-inducibility and light responsive element. Most NsGPXs contain the cis-acting elements related to plant hormones such as ABA, MeJA, GA, SA, and auxin, meaning they may portray roles in response to salt stress ( Figure 5).
According to the expression profiling of GPX genes, number of abiotic and biotic stresses have been revealed to induce the GPX genes. The qRT-PCR result shows that 5 NsGPXs have increasing levels of expression in N. sibirica leaf, stem and root from 24 h after salt stress until 48 h, which means NsGPXs may portray an important role in response to salt stress, especially in scavenging excessive ROS ( Figure 6). This result is also of considerable significance to further understand N. sibirica's mechanism of salt-tolerance. Still, the expression levels of NsGPXs in various tissues are significantly divergent, which is similar to the study on the Lotus japonicus GPX gene family [44]. The high expression of NsGPX1 in all plant parts under salt stress indicated that NsGPX1 was involved in the main process of salt stress resistance, while the significantly improved expression of NsGPX3 in leave after salt stress showed that its function may be similar to AtGPX3, which regulates REDOX status through H 2 O 2 , interacts with phospholipase ABI1/ABI2, activates plasma membrane Ca 2+ and K + channels, closes stomata, and participates in ABA and stress regulatory signal pathways [45]. In addition, though NtGPX3 and NtGPX4 are very similar in sequence composition and domain, which means they may have similar protein functions, their expression patterns are completely different under salt stress, indicating that the differentiation of their expression patterns can also affect the function of genes. Besides, we found no intraspecies collinearity in NsGPX gene family and no tandem repeat sequences through mcscanx, which means NtGPX3 and NtGPX4 are not duplicating genes ( Figure S3). This result differentiates from GPX gene family in cucumber, which the segmental along with tandem duplication has been revealed as the reliable source for the GPX gene family expansion [19]. Research conducted on Soybeans identified that GPX genes displayed substantial differential expression of such genes in response to oxidative, drought and salinity stresses in plant root tissue [46]. When N. sibirica roots under salt stress, the high expression of NsGPX7 revealed that its function in salt stress resistance may focus on the scavenging of excess ROS in roots. Related studies in A. thaliana have showed that both AtGPX1 and AtGPX7 are related to the reduction of H 2 O 2 under photooxidative stress, the functions of both genes are not completely redundant [47], which means depletion of GPX1 and GPX7 expression may affect the reduced tolerance to abiotic stress. Still, further research is needed to determine the molecular mechanism of NsGPXs function and to fully understand how exactly NsGPX gene family contributes to salt stress response by using various functional validation analyses, such as overexpression, knockout via CRISPR/Cas system, etc.

Conclusions
The current study identified a total seven NsGPX genes in N. sibirica via genome-wide analysis. For purpose of in-depth knowledge into the evolution of NsGPX gene family, gene structure, phylogenetic, conserved motifs, cis-elements, and expression profiling against salt stress treatments were accomplished. NsGPX genes expressed differentially in different tissues under salt stress, most of them have increasing levels of expression from 24 h after salt stress until 48 h, which means NsGPXs may play an important role in response to salt stress. These discoveries may lay a foundation for further research of NsGPXs in N. sibirica developmental processes and response to variable abiotic stresses, then help to further understand the salt resistance of N. sibirica, eventually aid in the discovery of new methods to restore overtly saline soil.

Summary
The current study filled the gap in related studies through the analysis of NsGPX gene family, and preliminarily explored the role of NsGPX in salt stress response, providing the basis for in-depth exploration stress response mechanism of N. sibirica. Our study is also fundamental to functional validation analyses, which is necessary to fully understand how exactly NsGPX gene family contributes to salt stress response. All these further studies will be the furtherance of utilization and ecological restoration of saline soil.
Supplementary Materials: The following supporting information can be downloaded at: https://www. mdpi.com/article/10.3390/genes14040950/s1, Figure S1: Electrophoresis gel plot of primer specificity detection and optimal Tm detection; Figure S2: The melting peaks of the qPCR results; Figure S3: Intraspecies collinearity analysis of NsGPXs. Table S1: The sequences of NsGPX genes; Table S2: qRT-PCR primers used to quantify NsGPX gene expression; Table S3: Prediction of subcellular localization of NsGPX proteins; Table S4: Types and numbers of known hormone-and stress-related cis elements in the promoter regions of NsGPX genes.