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Genome-Wide Characterization of Glutathione Peroxidase (GPX) Gene Family in Rapeseed (Brassica napus L.) Revealed Their Role in Multiple Abiotic Stress Response and Hormone Signaling

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. To understand the major roles of the GPX gene family in rapeseed (Brassica napus L.), for the first time, a genome-wide study identified 25 BnGPX genes in the rapeseed genome. The phylogenetic analysis discovered that GPX genes were grouped into four major groups (Group I–Group IV) from rapeseed and three closely interrelated plant species. The universal investigation uncovered that the BnGPXs gene experienced segmental duplications and positive selection pressure. Gene structure and motifs examination recommended that most of the BnGPX genes demonstrated a comparatively well-maintained exon-intron and motifs arrangement within the identical group. Likewise, we recognized five hormones-, four stress-, and numerous light-reactive cis-elements in the promoters of BnGPXs. Five putative bna-miRNAs from two families were also prophesied, targeting six BnGPXs genes. Gene ontology annotation results proved the main role of BnGPXs in antioxidant defense systems, ROS, and response to stress stimulus. Several BnGPXs genes revealed boosted expression profiles in many developmental tissues/organs, i.e., root, seed, leaf, stem, flower, and silique. The qRT-PCR based expression profiling exhibited that two genes (BnGPX21 and BnGPX23) were suggestively up-regulated against different hormones (ABA, IAA, and MeJA) and abiotic stress (salinity, cold, waterlogging, and drought) treatments. In short, our discoveries provide a basis for additional functional studies on the BnGPX genes in future rapeseed breeding programs.


Introduction
Plants are frequently exposed to several environmental stresses, comprising both abiotic and biotic, which significantly affect the growth and developmental processes [1][2][3][4]. These traumas can boost the production of reactive oxygen species (ROS), impairing cellular apparatuses and macromolecules, including DNA, proteins, and lipids, and eventually enquiry by e-value set to 1e −5 . The amino acid sequences of eight AtGPXs were retrieved from the TAIR Arabidopsis genome database (http://www.arabidopsis.org/, accessed on 1 July 2021) [31]. Moreover, a local software, HMMER 3.1 [32], was used for hunting the GPX genes with default constraints. Then, the HMM profile of the GPX domain (Pfam: PF00255) obtained as of the Pfam database (http://pfam.xfam.org/, accessed on 1 July 2021) was employed to detect GPX gene sequences. Finally, 25 BnGPX genes were recognized by merging the two methods in the rapeseed genome. Furthermore, we recognized GPX genes in diverse plant species, i.e., Brassica rapa and Brassica oleracea, and their genome sequences downloaded from the JGI Phytozome 12.0 database (https://phytozome.jgi.doe.gov/pz/ portal.html, accessed on 1 July 2021) [33] through a similar method.

Cis-Elements Analysis in the BnGPX Gene Promoters
To study the putative cis-elements in the BnGPXs promoters, we obtained the 2Kb sequence upstream of start codons in the Brassica napus genome. Then, the promoter sequence of the individual gene was examined using the PlantCARE website (http:// bioinformatics.psb.ugent.be/webtools/plantcare/html/, accessed on 1 July 2021) [42], and the figure was drawn using TBtools (V 1.068) [36].

Expression Analysis of BnGPX Genes in Diverse Tissues
For tissue-specific expression profiling, we downloaded RNA-seq data (BioProject ID: PRJCA001495, accessed on 1 July 2021) of rapeseed from the National Genomics Data Center. The complete method was described in a recent work [28]. Mainly, cuffquant and Cuffnorm were employed to create normalized counts in transcripts per million (TPM) values. Based on TPM estimates, the expression heat map was formed using GraphPad Prism 8 software (https://www.graphpad.com/, accessed on 1 July 2021) [45].

Plant Material and Stress Conditions
In the current study, a typical cultivated variety, ZS11, was used for stress treatments. The seeds of the ZS11 genotype were obtained from Wuhan Zhongyou Seed Technology Co., Ltd., Wuhan, China. The stress treatments were performed as designated in previous work [28]. Briefly, the vigorous seeds were carefully chosen and sterilized with 10% hypochlorous acid solution for 5 min. The seeds were grown on water-saturated filter paper in a chamber (25 • C day/night and 16 h/8 h light/dark cycle) until the radicle's extent turned up about 5 mm. For stress treatment, sprouted seeds were uncovered to 150 mM NaCl solution for salt stress, 15% PEG6000 solution for drought stress, and 4 • C for cold stress on water-saturated filter paper. For waterlogging stress, the seeds were flooded with water in the Eppendorf tube. To explore the influence of numerous hormones, the germinated seeds were planted in Murashige and Skoog (MS) medium supplied with 100 µM of each hormone such as abscisic acid (ABA), gibberellic acid (GA), methyl jasmonate (MeJA), and indole-acetic acid/auxin (IAA). The samples were collected at 0 (CK), 2, 4, 6, and 8 h after the treatments. Three biological repetitions were carried out for all the treatments. All the samples were rapidly frozen in liquid nitrogen and were kept at −80 • C for expression analysis.

RNA Extraction and qRT-PCR Analysis
Total RNA extraction and cDNA synthesis was performed using TransZol Up Plus RNA Kit (TransGen Biotech, Beijing, China) and cDNA Synthesis SuperMix (TransGen Biotech, Beijing, China) according to manufacturer instructions. The qRT-PCR was conducted with an ABI StepOne real-time fluorescence quantitative PCR instrument (Applied Biosystems, Foster City, CA, USA) using SYBR ® Green Supermix (Bio-Rad). The BnACTIN was used as an internal control. The qRT-PCR reaction was performed as follows: 94 • C for 10 min, followed by 40 cycles of 94 • C for 15 s, 60 • C for 30 s, and 72 • C for 10 s. Each qRT-PCR reaction was carried out with three biological triplicates, and the data were observed using the 2 −∆∆CT method [28,46]. All the primers used in this experiment are listed in Table S1. The heatmap was formed using GraphPad Prism 8 software [45].

Documentation and Characterization of GPX Gene Family in Rapeseed
Based on BLASTP examinations against the rapeseed genome employing eight Arabidopsis AtGPXs proteins as enquiries, a total of 25 presumed GPX genes were recognized in the complete rapeseed genome (Table 1). Henceforth, these genes are named "BnGPX1-BnGPX25", among which 13 genes (BnGPX1-BnGPX13) were located on the A subgenome, and 12 genes (BnGPX14-BnGPX25) were located on the C subgenome (Table 1). Comprehensive data of the 25 BnGPX genes is shown in Table 1. In brief, gene length varied from 1028 bp (BnGPX17) to 4654 bp (BnGPX9) with 5-6 exons in each sequence (Table 1). Eleven genes comprise six exons, and 14 genes consist of five exons ( Table 1). The CDS length varied from 363 bp (BnGPX10) to 726 bp (BnGPX23), whereas the protein length altered from 120 (BnGPX10) to 241 (BnGPX23) amino acids ( Table 1). The predicted molecular weights of the 25 BnGPX proteins ranged from 13.45 kDa (BnGPX10) to 26.97 kDa (BnGPX22), and the isoelectric points ranged from 5.18 (BnGPX24) to 9.58 (BnGPX22) ( Table 1). The subcellular localization results anticipated that 16 BnGPX proteins are located on the chloroplast, 6 proteins are located on the cytoplasm, 2 proteins (BnGPX3 and BnGPX9) are located on extracellular, and 1 protein (BnGPX20) is located on the nucleus (Table 1). Moreover, 8 GPX genes (BolGPX1-BolGPX8) from the Brassica oleracea and 12 GPX genes were also discovered from Brassica rapa (BraGPX1-BraGPX12) genomes (Table S2). In the genomic position, the positive (+) and negative (−) signs indicate the existence of a gene on the positive and negative strand of that specific marker, respectively.

Phylogenetic Relationships of GPX Genes
To discover the evolutionary history between the BnGPX, BolGPX, BraGPX, and AtGPX genes, an unrooted phylogenetic tree was assembled by a multiple sequence alignment (MSA) of the prophesied GPX protein sequences from Brassica napus, Brassica oleracea, Brassica rapa, and Arabidopsis thaliana, which was gathered into four main groups (Group I-Group IV) ( Figure 1). The findings revealed that Group I was comprised of 10 GPX members (4 BnGPXs, 2 BraGPXs, 2 BolGPXs, and 2 AtGPX); Group II was comprised of 17 GPX members (7 BnGPXs, 4 BraGPXs, 3 BolGPXs, and 3 AtGPX); Group III contained 17 GPX members (9 BnGPXs, 2 BolGPX, 4 BraGPXs, and 2 AtGPXs); and Group IV was comprised of 9 GPX members (5 BnGPXs, 1 BolGPXs, 2 BraGPXs, and 1 AtGPXs) ( Figure 1). Overall, GPXs clustering into the identical sub-group may possess parallel functions. It is worth mentioning that BnGPX genes were spread in each group with homologs from other plant species, and Group II and III were observed to have more BnGPXs, BolGPXs, BraGPXs, and AtGPXs members than the other three groups ( Figure 1). Additionally, it was noticed that the BnGPXs have a stronger phylogenetic link with the BolGPXs and BraGPXs in each group. 1). Overall, GPXs clustering into the identical sub-group may possess parallel functions. It is worth mentioning that BnGPX genes were spread in each group with homologs from other plant species, and Group II and III were observed to have more BnGPXs, BolGPXs, BraGPXs, and AtGPXs members than the other three groups ( Figure 1). Additionally, it was noticed that the BnGPXs have a stronger phylogenetic link with the BolGPXs and BraGPXs in each group.

Evaluation of Chromosomal Dispersal and Synteny Links of BnGPX Genes
The development of new gene family followers in plants' genome advancement is comparatively ascribed to tandem and segmental doubling events [47]. Thus, the corresponding events were explored in BnGPXs to clarify the rapeseed GPX gene duplication events. The chromosomal scatterings of 25 BnGPX genes were appraised. Notably, 8 out of the 19 chromosomes had BnGPX genes ( Figure 2). Concisely, chromosomes A03, A05, A07, A09, C03, C04, C06, and C08 had only one BnGPX gene ( Figure 2). Remarkably, the residual chromosomes did not have any BnGPX gene, and no tandem repeat paralogous genes were found in any regions. Moreover, eight paralogous genes were identified on

Evaluation of Chromosomal Dispersal and Synteny Links of BnGPX Genes
The development of new gene family followers in plants' genome advancement is comparatively ascribed to tandem and segmental doubling events [47]. Thus, the corresponding events were explored in BnGPXs to clarify the rapeseed GPX gene duplication events. The chromosomal scatterings of 25 BnGPX genes were appraised. Notably, 8 out of the 19 chromosomes had BnGPX genes ( Figure 2). Concisely, chromosomes A03, A05, A07, A09, C03, C04, C06, and C08 had only one BnGPX gene ( Figure 2). Remarkably, the residual chromosomes did not have any BnGPX gene, and no tandem repeat paralogous genes were found in any regions. Moreover, eight paralogous genes were identified on the A03, A05, A07, A09, C03, C04, C06, and C08 chromosomes ( Figure 2). These conclusions revealed that the duplication events played a critical role in developing the BnGPX family genes.
Antioxidants 2021, 10, x FOR PEER REVIEW 7 of 19 the A03, A05, A07, A09, C03, C04, C06, and C08 chromosomes ( Figure 2). These conclusions revealed that the duplication events played a critical role in developing the BnGPX family genes.  Table S3). In short, in the A subgenome, 7 Brassica napus genes exhibited syntenic links with 5 AtGPXs, 1 BraGPXs, and BolGPXs. In the C subgenome, 9 Brassica napus genes presented syntenic links with 4 AtGPXs, 3 BraGPXs, and 2 BolGPXs (Figure 3). Particularly, numerous homologues of Arabidopsis thaliana, Brassica rapa, and Brassica oleracea withstood a syntenic alliance with BnGPXs, implying that whole-genome duplication (segmental duplication) events portrayed a vital role in BnGPXs gene family evolution in the rapeseed genome (Table S3).  The Ka/Ks ratio is a substantial index in estimating the replication events and selection pressures [48]. Thus, to comprehend the evolutionary constraints on the BnGPX gene family, the Ka, Ks, and Ka/Ks ratio was evaluated. The results stated that all the repeated BnGPX gene pairs had a Ka/Ks ratio of <1 (Table S3); however, two gene pairs (BnGPX4/BraGPX4 and BnGPX7/BraGPX7) had a Ka/Ks ratio of >1 and experienced positive selection pressure (Table S3). Almost all genes experienced segmental duplication events. Similarly, Brassica rapa, Brassica oleracea, and Arabidopsis thaliana also experienced the purifying selective pressure (Table S3). Overall, the findings signify that the rapeseed GPX family genes might have encountered purifying and positive selection burden during the course of their evolution.

Evaluation of Gene Structures and Conserved Motifs of BnGPX Genes
The gene structures (exon-intron arrangements) of the BnGPX genes were examined to gain insight into the expansion of rapeseed GPX family genes. The results showed that introns of the BnGPXs ranged from 4-5 ( Figure 4A), and exons ranged from 5-6 in each sequence (Table 1). Group I contained 4 introns, whereas Groups II-IV include 4-5 introns. A similar trend was also observed for exons (5-6/gene) within the same groups ( Figure 4A; Table 1). Mainly, BnGPX2 and BnGPX15 have diverse gene structures in Group II. In short, Groups II, III, and IV exhibited analogous intron-exon arrangements, and only Group I had a distinct intron-exon arrangement. These outcomes stipulated that GPX members within a group astonishingly had the same gene structures, steady with their phylogenetic relatives.  Additionally, we examined the full-length protein sequences of 25 BnGPXs to distinguish their conserved motifs. The conserved motif of the BnGPX genes varied from 4-8. Overall, ten conserved motifs were recognized, and the motif dispersals were also analogous within the groups (Groups I-IV) ( Figure 4B; Table S4). For example, all members of Additionally, we examined the full-length protein sequences of 25 BnGPXs to distinguish their conserved motifs. The conserved motif of the BnGPX genes varied from 4-8. Overall, ten conserved motifs were recognized, and the motif dispersals were also analogous within the groups (Groups I-IV) ( Figure 4B; Table S4). For example, all members of Group I comprise 8; Group II contains 5-7; Group III includes 5-8; and Group IV encompasses 4-8 conserved motifs ( Figure 4B). Notably, only BnGPX10 had four motifs in Group IV ( Figure 4B). Interestingly, motif 7 was specific to Group I; motif 9 was specific to Group II; motif 10 was limited to two genes (BnGPX14 and BnGPX1) in Groups III and IV; and other motifs were nearly equally distributed on all the genes ( Figure 4B). In a nutshell, the group organization's evenness was strongly retained by exploring the conserved motifs dispersals, gene structures, and phylogenetic links, demonstrating that the GPX proteins have exceptionally preserved amino acid residues and members contained by a group might have analogous roles.

Cis-Elements in the Promoters of BnGPX Genes
To discriminate the gene functions and regulatory roles, cis-regulatory elements in BnGPXs promoter regions were inspected by searching a 2000 bp upstream region from each gene's transcriptional activation site against the PlantCARE database. The complete data of cis-elements are offered in Table S5. The results display that five phytohormone-correlated (auxin, (abscisic acid (ABA), gibberellin (GA), methyl jasmonate (MeJA), and salicylic acid (SA)) responsive elements comprising TCA-element, TGA-element, CGTCA-motif, ABRE, AuxRR-core, TGACG-motif, TATC-box, GARE-motif, P-box, etc., were documented ( Figure 5; Table S5). Predominantly, many phytohormone-connected elements were prophesied to be definite to some genes and extensively scattered ( Figure 5), suggesting the decisive role of these genes in phytohormone arbitration.
Additionally, four abiotic stress-accompanying (drought, low-temperature, anaerobic, and light) elements, comprising ARE, LAMP-element, LTR, MRE, TCCC-motif, GT1-motif, chs-CMA1a, AE-box, box 4, G-box, MBS, etc. were recognized ( Figure 5; Table S5). Primarily, several light-responsive elements were found to be extensively disseminated among all of the genes ( Figure 5; Table S5), suggesting the extensive role of BnGPXs against light stress. On the whole, outcomes directed that BnGPXs expression levels may depart under phytohormone and abiotic stress environments.

Genome-Wide Investigation of miRNA Targeting BnGPX Genes
Earlier reports demonstrated that miRNA-mediated regulation is associated with the stress responses in plants. Hence, to enhance our familiarity of miRNA-mediated posttranscriptional regulation on BnGPXs, the current study discovered five miRNAs targeting six BnGPX genes ( Figure 6A; Table S6). The miRNA-targeted sites are shown in Figure 6B, and the comprehensive data of all miRNAs targeted sites/genes are introduced in Table  S6. The outcomes disclosed that 4 members of the bna-miR164 family targeted four genes (BnGPX3, BnGPX4, BnGPX16, and BnGPX22); and one member of the bna-miR396 family targeted two genes (BnGPX13 and BnGPX20) ( Figure 6; Table S6). Primarily, BnGPX3, BnGPX4, BnGPX16, and BnGPX22 were projected to be targeted by a superior number of miRNAs ( Figure 6A; Table S6). The expression profiling of predicted miRNAs and their targeted sites/genes needs confirmation in the further investigation to oversee their biological functions in the rapeseed genome.

Figure 5. Evaluation of cis-regulatory elements in the BnGPXs promoters' regions that are linked with various hormoneand stress-responsive elements. Unique color boxes display unique identified cis-elements.
Additionally, four abiotic stress-accompanying (drought, low-temperature, anaerobic, and light) elements, comprising ARE, LAMP-element, LTR, MRE, TCCC-motif, GT1motif, chs-CMA1a, AE-box, box 4, G-box, MBS, etc. were recognized ( Figure 5; Table S5). Primarily, several light-responsive elements were found to be extensively disseminated among all of the genes ( Figure 5; Table S5), suggesting the extensive role of BnGPXs against light stress. On the whole, outcomes directed that BnGPXs expression levels may depart under phytohormone and abiotic stress environments.

Genome-Wide Investigation of miRNA Targeting BnGPX Genes
Earlier reports demonstrated that miRNA-mediated regulation is associated with the stress responses in plants. Hence, to enhance our familiarity of miRNA-mediated posttranscriptional regulation on BnGPXs, the current study discovered five miRNAs targeting six BnGPX genes ( Figure 6A; Table S6). The miRNA-targeted sites are shown in Figure  6B, and the comprehensive data of all miRNAs targeted sites/genes are introduced in Table S6. The outcomes disclosed that 4 members of the bna-miR164 family targeted four genes (BnGPX3, BnGPX4, BnGPX16, and BnGPX22); and one member of the bna-miR396 family targeted two genes (BnGPX13 and BnGPX20) ( Figure 6; Table S6). Primarily, BnGPX3, BnGPX4, BnGPX16, and BnGPX22 were projected to be targeted by a superior number of miRNAs ( Figure 6A; Table S6). The expression profiling of predicted miRNAs and their targeted sites/genes needs confirmation in the further investigation to oversee their biological functions in the rapeseed genome.

Functional Annotation Evaluation of BnGPX Genes
To further distinguish the BnGPX genes' roles, GO annotation and enrichment analysis were carried out based on biological process (BP), molecular function (MF), and cellular component (CC) classes. The BP, MF, and CC annotation conclusions presented abundant suggestively enriched terms (Table S7). For instance, the BP enrichment analysis uncovered that these genes were principally involved in response to hydrogen peroxide (GO:0042542), response to an organic substance (GO:0010033), cellular response to abiotic stimulus (GO:0071214), response to reactive oxygen species (GO:0000302), etc. (Table S7). Results of MF annotation revealed that these genes are involved in oxidoreductase activity (GO:0016684), antioxidant activity (GO:0016209), molecular_function (GO:0003674), glutathione peroxidase activity (GO:0004602), peroxidase activity (GO:0004601), etc. (Table S7). These terms also validate the function of BnGPX genes in ROS scavenging and antioxidant defense systems. The CC enrichment analysis confirms that these genes are mainly linked with cellular anatomical entity (GO:0110165), cytoplasm (GO:0005737), obsolete intracellular part (GO:0044424), obsolete cell part (GO:0044464), and organelle (GO:0043226), etc. (Table S7). Notably, few of these terms follow the prediction of subcellular localization of the GPX proteins. In short, CC, BP, and MF-GO annotation results proved the main role of BnGPXs in antioxidant defense systems, ROS, and response to stress stimulus.  Table S6 for the comprehensive data of all predicted miRNAs.

Functional Annotation Evaluation of BnGPX Genes
To further distinguish the BnGPX genes' roles, GO annotation and enrichment analysis were carried out based on biological process (BP), molecular function (MF), and cellular component (CC) classes. The BP, MF, and CC annotation conclusions presented abundant suggestively enriched terms (Table S7). For instance, the BP enrichment analysis uncovered that these genes were principally involved in response to hydrogen peroxide (GO:0042542), response to an organic substance (GO:0010033), cellular response to abiotic stimulus (GO:0071214), response to reactive oxygen species (GO:0000302), etc. (Table S7). Results of MF annotation revealed that these genes are involved in oxidoreductase activity (GO:0016684), antioxidant activity (GO:0016209), molecular_function (GO:0003674), glutathione peroxidase activity (GO:0004602), peroxidase activity (GO:0004601), etc. (Table  S7). These terms also validate the function of BnGPX genes in ROS scavenging and antioxidant defense systems. The CC enrichment analysis confirms that these genes are mainly linked with cellular anatomical entity (GO:0110165), cytoplasm (GO:0005737), obsolete intracellular part (GO:0044424), obsolete cell part (GO:0044464), and organelle (GO:0043226), etc. (Table S7). Notably, few of these terms follow the prediction of subcellular localization of the GPX proteins. In short, CC, BP, and MF-GO annotation results proved the main role of BnGPXs in antioxidant defense systems, ROS, and response to stress stimulus.  Table S6 for the comprehensive data of all predicted miRNAs.

Tissue-Specific Expression Profiles of BnGPX Genes in Rapeseed
The tissue-specific expression profiles of BnGPXs genes were observed in six diverse tissues and organs, i.e., roots, stems, leaves, flowers, seeds, and silique, using RNA-seq data from Brassica napus (ZhongShuang 11 variety) (BioProject ID PRJCA001495). The expression profiles of the BnGPX genes altered in the different tissues and organs. As illustrated in Figure 7, group II genes exhibit higher expression in all of the tissues except BnGPX8, BnGPX12, and BnGPX18, which displayed relatively lower expression in seeds (Figure 7). On the other hand, group I and III genes show relatively lower expression in all of the tissues except BnGPX2, BnGPX4, BnGPX15, and BnGPX22 that showed somehow higher expression in leaf, flower, seeds, and silique ( Figure 7). Thus, it is noteworthy that group II genes may play substantial roles in rapeseed developmental processes. pression profiles of the BnGPX genes altered in the different tissues and organs. As illustrated in Figure 7, group II genes exhibit higher expression in all of the tissues except BnGPX8, BnGPX12, and BnGPX18, which displayed relatively lower expression in seeds (Figure 7). On the other hand, group I and III genes show relatively lower expression in all of the tissues except BnGPX2, BnGPX4, BnGPX15, and BnGPX22 that showed somehow higher expression in leaf, flower, seeds, and silique ( Figure 7). Thus, it is noteworthy that group II genes may play substantial roles in rapeseed developmental processes.
showed considerable expression under ABA, IAA, salinity, waterlogging, and drought conditions at certain time points (Figure 8). In comparison, the rest of the genes showed lower expression under all of the stress conditions. The results signify that mainly BnGPX21 and BnGPX23 genes could play a vital role in alleviating the harmful impact of different hormones and abiotic stress conditions in rapeseed.

Discussion
Rapeseed is an allotetraploid crop that practices widespread genome replication and integration activities [49]. However, rapeseed yield is influenced by numerous abiotic pressures, including temperature (cold/heat), high salinity, drought, waterlogging, and heavy metals toxicity [24,27,50]. The generation of ROS in response to normal and stress atmospheres is assessed and scavenged via GPXs, which is considered one of the most effective ROS antioxidant scavenging enzymes [6,9]. Further, GPX enzyme and GPX genes also contribute to several plant physiological, biochemical, and molecular mechanisms to withstand harsh environmental stresses [5,6]. In the recent past, GPX gene families have been reported in numerous crop plants, including five GPX genes in date palm (Phoenix dactylifera L.) [51] and in Rhodiola crenulate [52]; six genes in cucumber (Cucumis sativus L.) [53], watermelon (Citrullus lanatus L.) [54], and Lotus japonicus [55]; seven genes in sorghum (Sorghum bicolor L.) [19]; eight genes in Thellungiella salsuginea [56] and peach fruit (Prunus persica L.) [57]; 13 genes in cotton (Gossypium hirsutum L.) [58]; and 14 genes in bread wheat [59]. To the best of our knowledge, the GPX gene family was yet to be reported in rapeseed that responds to multiple stress conditions. The availability of the entire rapeseed genome justifies the genome-wide description of the GPX gene family, which could be used for forthcoming rapeseed breeding programs.

Discussion
Rapeseed is an allotetraploid crop that practices widespread genome replication and integration activities [49]. However, rapeseed yield is influenced by numerous abiotic pressures, including temperature (cold/heat), high salinity, drought, waterlogging, and heavy metals toxicity [24,27,50]. The generation of ROS in response to normal and stress atmospheres is assessed and scavenged via GPXs, which is considered one of the most effective ROS antioxidant scavenging enzymes [6,9]. Further, GPX enzyme and GPX genes also contribute to several plant physiological, biochemical, and molecular mechanisms to withstand harsh environmental stresses [5,6]. In the recent past, GPX gene families have been reported in numerous crop plants, including five GPX genes in date palm (Phoenix dactylifera L.) [51] and in Rhodiola crenulate [52]; six genes in cucumber (Cucumis sativus L.) [53], watermelon (Citrullus lanatus L.) [54], and Lotus japonicus [55]; seven genes in sorghum (Sorghum bicolor L.) [19]; eight genes in Thellungiella salsuginea [56] and peach fruit (Prunus persica L.) [57]; 13 genes in cotton (Gossypium hirsutum L.) [58]; and 14 genes in bread wheat [59]. To the best of our knowledge, the GPX gene family was yet to be reported in rapeseed that responds to multiple stress conditions. The availability of the entire rapeseed genome justifies the genome-wide description of the GPX gene family, which could be used for forthcoming rapeseed breeding programs.
According to the available literature, the current study identified the largest GPX gene family (25 BnGPXs) in the rapeseed genome (Table 1), which were grouped into four major groups (Figure 1). Deviations in the GPXs gene numbers between different plant species may be attributed to gene replication experiences, involving tandem and segmental replications, and participate in spreading GPXs for deviation. Gene replication of GPX genes was also uncovered in different plant species [19,57,58]. The current study showed that BnGPXs underwent segmental duplication, purifying and positive selection burden during their evolution (Table S3). Subsequently, these consequences recommended that BnGPXs replication activities might play an imperative role in gene evolution.
The phylogenetic tree showed that GPXs genes from rapeseed and other three plant species, including Brassica rapa, Brassica oleracea, and Arabidopsis thaliana, were categorized into four main groups (Figure 1), which was constant with the grouping in other plant species such as Thellungiella salsuginea [56], cotton [58], sorghum [19], and bread wheat [59]. Fascinatingly, each group comprised one or more GPXs genes from all plant species, and every BnGPX was extremely correlated to its homologs in the other three plant species, particularly in Brassica rapa and Brassica oleracea, suggesting that the mutual ancestor was self-possessed of pairs of possible orthologs. Additionally, gene structure analysis discovered that most of the BnGPX genes owned five introns ( Figure 4; Table 1); however, the number of exons ranged widely from four to five, and introns number varied from five to six in each group (Figure 4). Similar findings were also reported in cucumber, where all GPX genes had four to five introns and four to five exons [53]. In cotton, six exons and five introns were found in 13 GPX genes [58]. The exon-intron gatherings' divergence was practiced by three essential methods (exon/intron-gain/loss, exonization/pseudoexonization, and insertion/deletion), and they are precisely supported to structural divergence [60]. Remarkably, the GPX genes in each group offered parallel exon-intron organization and conserved motifs (Figure 4), indicating that these genes may take part in the matching roles associated with multiple abiotic cues. Similar discoveries have also been described in Thellungiella salsuginea [56], cotton [58], sorghum [19], and bread wheat [59].
To better comprehend the role of the BnGPX genes in contradiction of some environmental factors, cis-elements in the promoter regions were predicted. The outcomes demonstrated that two major kinds of cis-elements were identified, i.e., stress-and phytohormoneresponsive ( Figure 5; Table S5). Almost all recognized cis-elements were allied with phytohormones (ABA, MeJA, GA, SA, and auxin), and abiotic stress (drought, low-temperature, light, and anaerobic induction) ( Table S5). The previous report suggests that cis-elements support plant stress responses [61]. Moreover, these gene functions were additionally deeprooted by the GO annotation investigation (Table S7). Our results are in agreement with previous reports, where GPX genes contributed to several stress responses [19,51,55,56,59]. These conclusions can increase our comprehension of BnGPX genes under varied environmental circumstances.
In recent years, ample miRNAs have been documented by genome-wide investigation in rapeseed to take part in various environmental issues [62][63][64][65][66]. None of the previously reported GPXs gene families in different plants has reported miRNA-targeting GPX genes. Therefore, for the first time, the current study discovered five miRNAs from two families (miR164 and miR396) targeting six BnGPX genes ( Figure 6; Table S6). In a recent study, Vm-milR37 subsidized in pathogenicity through regulating the VmGPX gene, which takes part in the oxidative stress response throughout Valsa mali infection [67]. Notably, there is no report on miRNAs targeting GPX genes in plants. However, the identified miRNAs have been documented in different plants and stress conditions. For instance, miR164 regulated the salinity stress tolerance mechanism in maize (Zea mays L.) [68]. In another study, miR164-targeted NAC genes undesirably regulated drought stress tolerance in rice plants [69]. Further, miR164 negatively regulated the resistance of wheat to stripe rust [70]. Similarly, another miRNA (miR396) arbitrated amendment in plant growth and salt stress responses in creeping bentgrass (Agrostis stolonifera) [71]. Further, miR396 was found to be elaborated in plant response to vernalization and flower advancement in Agrostis stolonifera [72]. In another study, the overexpression of osa-MIR396c declines salinity and alkali stress tolerance in rice [73]. It is worth mentioning that these findings support our outcomes and endorse that bna-miRNAs might play critical roles in the contradiction of various stresses by fluctuating the transcript profiles of GPX genes in rapeseed.
Growing evidence specifies that GPXs genes may display diverse expression profiles in different organs/tissues and under stress circumstances [19,51,55,56,59]. Hence, the tissuespecific expression profiles of BnGPXs genes were evaluated in six diverse developmental tissues using RNA-seq data (Figure 7), and these results are in agreement with previous reports. For example, six watermelon ClGPX genes presented higher expressions in flowers and fruits [54]. In cotton, almost all GhGPXs genes exhibited the uppermost expression in flowers [58]. In cucumber, some CsGPX genes showed higher expression in roots and flowers [53]. The innumerable expression profiles of BnGPX genes (mainly from Group II) specify that they may play diverse purposeful roles at certain developmental procedures in rapeseed.
Similarly, the expression profiles of eight BnGPX genes were appraised under diverse phytohormones and abiotic stress treatments (Figure 8). Among all evaluated genes, BnGPX21 and BnGPX23 showed higher expression under almost all stress treatments except GA (Figure 8). These consequences agreed with earlier discoveries where several GPX genes exhibited sophisticated expression against stress treatments. For example, in watermelon, almost all ClGPX genes were up-regulated in response to cold, drought, and salinity stresses [54]. Nearly all T. salsuginea TsGPXs genes were suggestively up-regulated at a one-time point under salinity and drought treatments [56]. Pennisetum glaucum PgGPX expression levels were highly induced by salinity and drought conditions [12]. In cucumber, almost all six CsGPX genes were significantly up-regulated at some time points against cold, salinity, drought, and ABA treatments [53]. These discoveries offered robust evidence that GPX genes play a well-maintained role in defending abiotic and hormone stresses in diverse plant species.

Conclusions
In conclusion, a total of 25 BnGPX genes were identified in the rapeseed genome. To gain in-depth insights into the evolution of the GPX gene family in rapeseed genome, gene structure, phylogenetic and synteny, conserved motifs, cis-elements, GO annotation, miRNA prediction, tissue-specific expression, and expression profiling against diverse hormones, and abiotic stress treatments were carried out. The BnGPX genes were differentially expressed in numerous tissues/organs, signifying that these genes might contribute precise roles in rapeseed development. The outcomes of gene expression profiling in response to diverse abiotic stresses and hormones treatments discovered that several BnGPX genes might contribute to stress responses and hormone signaling pathways. These discoveries will lay the basis for studying the roles of BnGPX genes in rapeseed developmental processes and response to several stresses using different functional validation options, such as overexpression, knockout via CRISPR/Cas system, etc.
Supplementary Materials: The following are available online at https://www.mdpi.com/article/ 10.3390/antiox10091481/s1, Table S1: A list of primers used for gene expression analysis in qRT-PCR, Table S2: The sequence of GPX family genes in Brassica napus, Arabidopsis thaliana, Brassica oleracea, and Brassica rapa, Table S3: The data of gene duplication type, Ka, Ks, and Ka/Ks ratio values of B. napus and other three plant species, Table S4: Information on the 10 identified motifs in BnGPX proteins, Table S5: Information on hormone-and stress-related cis-elements identified in the promoters regions of BnGPXs, Table S6: Information on miRNA targeted BnGPX genes, Table S7: The GO enrichment analysis of BnGPX genes.

Data Availability Statement:
The datasets used and/or analyzed during the current study are shown in the supplementary files. For tissue-specific expression profiling, we downloaded RNA-seq data of rapeseed (BioProject ID: PRJCA001495, accessed on 1 July 2021) from the National Genomics Data Center. The rapeseed genome sequence was downloaded from BnPIR database (http: //cbi.hzau.edu.cn/bnapus/index.php, accessed on 1 July 2021). The sequences of Brassica rapa and Brassica oleracea are available at Phytozome database (https://phytozome.jgi.doe.gov/pz/portal.html, accessed on 1 July 2021).

Conflicts of Interest:
The authors declare no conflict of interest.