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Article

Genome-Wide Identification and Characterization of the Superoxide Dismutase (SOD) Gene Family in Pakchoi and the Role of the BchFSD2 Gene in the Salt Stress Toleran

by
Yuqi Zhou
,
Shuhao Li
,
Shengxiang Ran
,
Yang Xu
,
Maomao Hou
,
Mingxuan Han
and
Fenglin Zhong
*
College of Horticulture, Fujian Agriculture and Forestry University, Fuzhou 350002, China
*
Author to whom correspondence should be addressed.
Agronomy 2024, 14(2), 384; https://doi.org/10.3390/agronomy14020384
Submission received: 26 January 2024 / Revised: 13 February 2024 / Accepted: 13 February 2024 / Published: 16 February 2024
(This article belongs to the Section Crop Breeding and Genetics)
Browse Figures
Versions Notes

Abstract

:
Superoxide dismutase (SOD) is an important antioxidant metalloenzyme present in plants that plays a vital role in plant growth and development, but studies on the SOD gene family in Pakchoi are lacking. In this study, we identified 13 SOD genes from pakchoi, including three MnSODs, five Cu/ZnSODs, and five FeSODs. Through structural analysis, the gene structures and motif patterns in the three subfamilies showed a high degree of conservation. From an evolutionary point of view, gene duplication is an important pathway driving the evolutionary development of the SOD gene family. In addition, by analyzing the structure and function of BchSOD proteins, most of the genes were shown to be involved in different developmental stages of pakchoi, and their expression was shown to be regulated by external conditions such as light, phytohormones, and abiotic stress. qPCR results revealed that BchSODs were expressed in different parts of pakchoi, and most of the genes were expressed in response to abiotic stresses (salt and drought) and hormones (GA and MeJA). In addition, the BchFSD2 gene was studied in depth, and subcellular localization confirmed that the BchFSD2 gene was expressed in plant chloroplasts. Overexpression of BchFSD2 promoted salt tolerance, limited superoxide anion and MDA production, and increased antioxidant enzyme activities in Arabidopsis. In summary, the BchSOD gene family was comprehensively analyzed in this study to provide new insights for a better understanding of BchSOD function and to improve salt tolerance in pakchoi.

1. Introduction

Plants live in ever-changing environments that typically have periods of unfavorable or stressful conditions that affect plant growth and development. These negative environmental situations involve biotic stresses, such as predator attacks, as well as abiotic stresses, such as heat, nutrient deficiencies, drought, cold, and an overabundance of salts [1]. Plant life processes are bound to involve reactive oxygen species (ROS) production. ROS consist of oxygen radicals such as superoxide (O2. −), hydroxyl (OH), and other nonradial species such as hydrogen peroxide (H2O2) [2]. However, various biotic and abiotic stressors can influence ROS homeostasis. In addition, many detrimental effects can result in excessive accumulation of ROS, causing oxidative stress, which can lead to damage to essential cellular components, including nucleic acids, proteins, carbohydrates, and lipids, and may ultimately cause programmed cell death [3,4].
In an attempt to defend themselves against excess ROS due to deleterious effects, plant cells and their organelles employ antioxidant mechanisms. Cellular antioxidant mechanisms are among the major ways to defend against various types of stresses [4]. The plant antioxidant defense system includes numerous nonenzymatic and enzymatic antioxidants. The principal nonenzymatic antioxidants consist of ascorbic acid (ASA), glutathione (GSH), and phenolic compounds. On the other hand, enzymatic antioxidants include superoxide dismutase (SOD), catalase (CAT), ascorbate peroxidase (APX), and glutathione reductase (GR) [5]. Among the many known antioxidant enzymes, SOD is commonly found in all aerobic organisms and in all subcellular cells susceptible to ROS-mediated oxidative stress, and it is one of the most potent components of the antioxidant defense against ROS toxicity in plant cells. It has been shown that O2.− is eliminated by SOD-catalyzed disproportionation, where one O2.− is converted into H2O2 and the other into O2 [4]. According to the metal cofactors, plant SODs include four main types: FeSODs (iron cofactors), MnSODs (manganese cofactors), Cu/ZnSODs (with Cu and Zn serving as cofactors, where Cu is the redox-active catalytic metal), and NiSODs (nickel cofactors). To date, no NiSOD has been identified in plants [6].
In recent years a large number of researchers have shown that the SOD gene plays a crucial position in plant resistance to harsh environmental conditions and is responsive to a vast array of environmental signals. For instance, several studies have identified SODs as pivotal genes that mediate immunity to heavy metal toxicity in tobacco [7]. Shiraya [8] et al. identified MSD1 as a unique Golgi somatic type of SOD in heat-tolerant rice varieties (Oryza sativa) that enhances the ability of plants to adapt to heat. In addition, Mosa [9] et al. reported that the expression of the Cu-ZnSOD gene was induced in cucumber plants (Cucumis sativus) by copper nanoparticles. Taken together, the results of these studies indicate that plant stress tolerance can be enhanced by increasing SOD activity and the expression of SOD-encoding genes. To date, genome-wide identification and studies of the SOD gene family have been carried out in a number of plants, including Nicotiana tabacum [7], rapeseed [10], tomato [11], Larix kaempferi [12], Salvia miltiorrhiza [13], and grapevine [14].
Pakchoi (Brassica campestris L. ssp. Chinensis) is a leafy vegetable widely grown in Asian countries. To date, genome-wide characterization of the SOD gene family in pakchoi has not been reported. In this study, we identified the SOD gene family in pakchoi at the genome-wide level and systematically analyzed its physicochemical properties, gene structure, homology, and phylogenetic developmental relationships. The cis-acting elements of the promoter region of the BchSOD genes were also predicted and analyzed. In addition, the BchSOD genes were analyzed for tissue-specific expression as well as for expression patterns in response to abiotic stresses (salt and drought) and hormone treatments (gibberellin and methyl jasmonate). We also performed subcellular localization and constructed an overexpression vector of the abiotic stress-sensitive gene BchFSD2 to investigate further its function and regulatory network under salt stress. This study lays the foundation for further research on the function of the SOD gene in pakchoi under different external influences.

2. Materials and Methods

2.1. Genome-Wide Identification of SOD in Pakchoi

In this study, we used BLASTP and the Hidden Markov Model to identify SOD genes in pakchoi. The whole pakchoi genomic dataset, which included coding sequence (CDS), genetic feature format (GFF), and protein sequence information, was previously evaluated by our laboratory and downloaded from the figshare database (https://figshare.com/articles/dataset/Thegenomeannotationinformationofpakchoi/19589524 (accessed on 1 March 2023)). The protein sequences of the SODs described in the model plant Arabidopsis were downloaded from the NCBI database (https://www.ncbi.nlm.nih.gov/gene/ (accessed on 1 July 2023)). Using the Arabidopsis SOD protein sequence as the query sequence, the BLAST Zone program of Tbtools was used to search for the pakchoi SOD protein. Furthermore, all the predicted pakchoi SOD family members were confirmed through Pfam (http://pfam.xfam.org/ (accessed on 1 July 2023)) [15]. The physicochemical parameters, including length, molecular weight, isoelectric point, GRAVY, and instability index, were analyzed by the online tool ExPASy server (https://web.expasy.org/ (accessed on 1 March 2023)) [16]. Tbtools was used to map the SOD genes to the pakchoi chromosomes [17], and the subcellular location of each protein was investigated by WoLF PSORT (https://wolfpsort.hgc.jp/ (accessed on 1 July 2023)).

2.2. Phylogenetic Analysis of BchSOD Genes

The SOD amino acid sequences of different plant species, such as Brassica rapa and Brassica oleracea, were downloaded from the JGI Phytozome 12.0 database (https://phytozome-next.jgi.doe.gov/ (accessed on 2 July 2023)) [10]. For further classification, we constructed a phylogenetic tree of the B. rapa, B. oleracea, B. chinensis, and A. thaliana protein sequences. The sequences were aligned using MEGA7.0 software, and a phylogenetic tree was constructed using the neighbor-joining (NJ), maximal and minimal method with a 1000 bootstrap and other parameters set to default values [18]. Then, the Evolview v3 website (https://www.evolgenius.info/evolview/, (accessed on 2 July 2023)) [19] was used to visualize the phylogenetic tree.

2.3. Analysis of BchSOD Gene Structures, Conserved Motifs, and Conserved Domains

The BchSOD gene structures were analyzed using Tbtools V2.042 software. The MEME tool (https://meme-suite.org/meme/tools/meme, (accessed on 3 July 2023)) was used to predict the conserved motifs, with the motif value set to ten, and the site distribution set for any number of repetitions and other parameters at default. Then, Tbtools V2.042 software was used to visualize the conserved motifs.

2.4. Chromosomal Localization and Synteny Analysis of BchSOD Genes

The chromosomal distribution and collinearity mapping of the BchSOD genes were performed using the advanced circos module in Tbtools [17]. Then, synteny analysis of the SOD genes was performed with the One Step MCScanX module of Tbtools from B. rapa, B. oleracea, B. chinensis, and A. thliana.

2.5. Cis-Acting Regulatory Elements in Promoter Analysis of BchSOD Genes

The promoter sequences of the Pakchoi BchSOD genes (2000 bp upstream) were obtained by the Gtf/Gff3 Sequence Extraction module of Tbtools. The online tool PlantCARE (http://bioinformatics.psb.ugent.be/webtools/plantcare/html/ (accessed on 5 July 2023)) was used to analyze the cis-acting regulatory elements.

2.6. Prediction of the 2D and 3D Structure of BchSOD Proteins

The secondary structure of the BchSOD proteins was predicted using the Prabi website (Available online: https://npsa-prabi.ibcp.fr/cgi-bin/npsa_automat.pl?page=/NPSA/npsa_sopma.html (accessed on 12 July 2023)). The predicted tertiary structures of BchSOD proteins were visualized through the SWISS-MODEL website (Available online: https://www.swissmodel.expasy.org/ (accessed on 12 July 2023)) with default parameters [20].

2.7. Plant Materials and Growth Conditions

In this study, the hybrid pakchoi variety ‘Jinpin Hanchun’ (Jinpin Agriculture, Fujian, China) was used as a test material, and experimental treatments were carried out. After the seeds were soaked and germinated, the uniformly growing plants were selected for hydroponics with Hoagland nutrient solution and cultured under a photoperiod of 12 h of light with a light intensity of approximately 180 ± 15 μmol·m−2·s−1 PPFD at a temperature of 25 ± 3 °C. The plants were treated when four leaves and one heart were present. To simulate salt and drought conditions, 150 Mm Nacl [10] and 10% PEG6000, respectively, were added to the culture solution. Tender leaves were collected from treated plants in three independent biological replicates at 0 h, 12 h, 24 h, 36 h, and 48 h after each treatment. For phytohormone treatments, leaves were sprayed once with freshly prepared working solutions of 100 μM GA [10] or 100 μM MeJA. Tender leaves were collected from treated plants in three independent biological replicates at 0 h, 2 h, 4 h, 6 h, and 8 h after each treatment. Finally, all the samples were packed in aluminum foil, immediately submerged in liquid nitrogen, and stored at −80 °C until further analysis.

2.8. BchSOD Gene Expression Pattern Analysis in Different Tissues

To analyze the specific expression of the BchSOD genes in different tissues, five leaves and one heart, roots, petioles, cotyledons, and true leaves of ‘Jinpin Hanchun’ pakchoi plants hydroponically cultivated in Hoagland nutrient solution were selected as experimental materials. The samples were frozen in liquid nitrogen and then stored at −80 °C.

2.9. RNA Extraction and qRT-PCR Analyses

Total RNA extraction and cDNA synthesis were performed using a FastPure Plant Total RNA Isolation Kit (Vazyme Biotech, Nanjing, China) and FastKing gDNA Dispelling RT SuperMix (TranGen Biotech, Beijing, China) according to the manufacturer’s instructions. qRT-PCR was performed on a Roche LightCycler 96 PCR system, following the recommended guidelines for 2×RealStar Green Fast Mixture (GenStar Biotech, Beijing, China). The primers for qPCR were designed using Primer Premier 5.0 and are listed in Supplementary Table S1. The actin gene of pakchoi was used as an internal control. Three biological replicates were carried out for each cDNA sample. The cycle threshold (CT) data that were obtained were calculated using the 2−△△CT method to determine the relative expression of the BchSOD genes [21]. Finally, heatmaps were created using Tbtools V2.042 software.

2.10. Subcellular Localization of BchFSD2

To further determine the subcellular localization of the BchFSD2 gene, the protein-coding region of the target gene was amplified using Phanta Max Super-Fidelity DNA Polymerase (Vazyme Biotech, Nanjing, China). The primers for PCR were designed using Primer Premier 5.0 and are listed in Supplementary Table S2. The CDS of BchFSD2 was cloned and inserted into the PGWB605 vector by the Gateway method using a gateway kit (Thermo Fisher Scientific, Shanghai, China) to generate the C-terminal fusion green fluorescent protein (GFP) construct 35: BchFSD2-GFP. This construct was subsequently transformed into Agrobacterium tumefaciens receptor GV3101 cells (HeRui, Fujian, China). The injection method was used for transient expression in tobacco leaves, after which the plants were subjected to darkness for 24 h and subsequently incubated under normal conditions for 48 h. Then, fluorescence was observed under a confocal microscope (Olympus Fv1200, Shanghai, China).

2.11. Transformation of Arabidopsis Thaliana Overexpressing the BchFSD2 Gene

The recombinant Agrobacterium tumefaciens plasmid harboring the target gene BchFSD2 was transformed into wild-type Arabidopsis thaliana using the floral dip method [22]. After the T0 generation was harvested, the plants were sterilized and sown in 1/2 MS medium supplemented with 10 mg/L glufosinate-ammonium antibiotic, and when four true leaves were growing and normal-growing Arabidopsis plants were regarded as positive seedlings, they were transplanted into nutrient soil and considered the T1 generation, and the plants were subjected to PCR. Subsequently, the procedure was repeated until the pure strain of the T3 generation was obtained. Finally, the overexpression efficiency of the T3 pure strain BchFSD2 was verified by qRT-PCR.

2.12. Abiotic Stress Treatment and Related Physiological Index Detection

Transgenic Arabidopsis thaliana (L1) and wild-type Arabidopsis (WT) plants were cultured in ½ MS medium until four leaves were reached (12 days) and subsequently transferred to soil for 11 days for salt stress and drought stress treatments. For the salt stress treatments, L1 and WT plants were watered with 150 Mm Nacl at 3 d intervals, while the transgenic Arabidopsis control (L1-CK) and wild-type control (WT-CK) plants were watered at the same time. Samples were taken after 9 days of salt treatment for physiological data determination.
Physiological indices were mainly determined for hydrogen peroxide (H2O2), superoxide anion, and malondialdehyde (MDA) contents as well as superoxide dismutase (SOD), peroxidase (POD) and catalase (CAT) activity assays in experimental (L1 and WT) and control (L1-CK and WT-CK) cabbages under salt stress. The H2O2 content was determined using the H2O2 assay kit (Keming, Suzhou, China). About 0.15 g of leaves were ground and lysed in 1 mL of lysing solution, followed by the addition of reaction solution according to the instructions of the kit for the reaction. The absorbance was measured at 415 nm, and then the standard curve was used in order to calculate the content of H2O2. Superoxide anion and MDA contents, as well as SOD, POD, and CAT activities, were also determined using the corresponding assay kits (Keming, Suzhou, China), respectively. Tissue extraction was performed as previously described, with specific experiments also following the instructions provided with the kits, and three technical replicates were performed for each treatment.

3. Results

3.1. Identification and Characterization of the SOD Gene Family in Pakchoi

Based on the results of the HMM search and BLASTP, 13 BchSOD genes were found in pakchoi. According to structural domain analysis, the numbers of proteins with a Mn-SOD domain (Pfam:02777), a Cu/Zn-SOD domain (Pfam: 00080), and a Fe-SOD domain (Pfam:00081) were 3, 5, and 5, respectively. Therefore, based on their species name and Pfam types, these genes were named BchMSD1-BchMSD3, BchCSD1-BchCSD5, and BchFSD1-BchFSD5, respectively (Table 1). The physical and chemical characteristics of the 13 BchSODs are shown in Table 1. All 13 BchSOD genes were located in the A subgenome. The protein and CDS lengths and molecular weights were 152–299 amino acids, 459–900 bp, and 37.83–73.4 kDa, respectively. Moreover, the instability exponents of the BchSOD proteins varied from 41.68 to 66.86; the isoelectric point varied from 5.08 to 5.21, indicating that all the members of BchSOD are acidic; and the GRAVY values ranged from 0.684 to 1.017, indicating that all the BchSODs are hydrophobic proteins. The subcellular localization results revealed that six proteins (BchCSD3, BchCSD4, BchFSD1, BchFSD3, BchFSD4, and BchFSD5) were predicted to localize to the chloroplast, three proteins (BchMSD2, BchMSD3, and BchFSD2) were located on the mitochondria, three proteins (BchCSD1, BchCSD2, and BchCSD5) were located in the cytoplasm, and the remaining protein (BchMSD1) was located in the extracellular region space (Table 1).

3.2. Phylogenetic Analysis of SOD Genes

We constructed a phylogenetic tree by comparing the full-length sequences of SOD proteins from A. thaliana, B. oleracea, B. napus, and B. chinensis to determine the evolutionary relationship of SOD in different species. The 66 SODs were categorized into three classes according to their structural domain and phylogenetic tree (Figure 1 and Figures S2 and S3). The results indicated that the Cu/Zn-SOD group included twenty-eight SOD members (six BolSODs, fourteen BnSODs, five BchSODs, and three AtSODs), the Mn-SOD group included fourteen SOD members (three BolSODs, six BnSODs, three BchSODs, and two AtSODs), and the Fe-SOD group included twenty-four SOD members (five BolSODs, eleven BnSODs, five BchSODs, and three AtSODs). Moreover, BchCSD5 and BnCSD7, BchCSD2 and BnCSD6, BchCSD1 and BolCSD2, BchMSD2 and BnMSD1, BchMSD3 and BolMSD1, BchMSD1 and BnMSD3, BchFSD1 and BnFSD2, BchFSD2 and BolFSD4, BchFSD5 and BnFSD4, BchFSD4 and BolFSD3 were the smallest branches, respectively; furthermore, the BchSODs were closer to the BolSODs and BnSODs during the evolutionary process.

3.3. Conserved Motifs and Gene Structures Analysis of BchSOD Genes

To understand the structural variation of the BchSOD proteins and determine their functions, we analyzed the conserved motifs of the BchSOD genes using the MEME tool and visualized them with TBtools (Figure 2B and Table S3). The results revealed that 10 motifs were present in BchSODs (Figure 2D). The conserved motifs of the BchSOD proteins varied from two to seven, and the analysis revealed that similar related members in the same subfamily had the same motif formation. Motifs 1,4, and 3 were predicted in all the Fe-SOD and Mn-SOD members. Motif 5 was predicted for most proteins of the Fe-SOD and Cu/Zn-SOD genes, except for BchCSD3, BchFSD1, and BchFSD2. Interestingly, motifs 2 and 10 existed only in Cu/Zn-SOD subfamily members, motif 9 was a unique conserved motif in Mn-SOD subfamily members, and motifs 6 and 8 existed only in Fe-SOD subfamily members. Further examination of the motif logos plot (Figure 2D) revealed that motifs 6, 7, and 9 were more conserved. Therefore, according to our predictions, the members of the Fe-SOD and Mn-SOD subgroups were more conserved.
The study of gene structure has aided in understanding the evolution of the SOD family of genes in pakchoi. The results demonstrated that the quantities of exons and introns in the BchSOD genes were 5–9 and 5–8, respectively (Figure 2C). The Mn-SOD subfamily includes six exons and five introns. The Cu/Zn-SOD group had 6 to 7 exons and 5 to 8 introns. The Fe-SOD group contained eight exons and seven introns, except for BchFSD1, which contained nine exons and eight introns. Notably, the BchMSD2 and BchMSD3 genes exhibited similar gene structures. In summary, the BchSOD genes differed significantly in terms of the number of exons and introns but exhibited similar intron/exon patterns within the same evolutionary branch.

3.4. Analysis of Cis-Acting Elements in Promoters of BchSOD Genes

SOD genes play a crucial role in the response to external stresses. We analyzed cis-acting elements in the promoters of BchSOD genes by using 2000 bp upstream regions to investigate hypothetical cis-acting elements that play critical roles in stress, light, and hormone modulation. Overall, 314 cis-acting elements were identified in the BchSOD genes (Figure 3), and additional detailed information on the cis-acting elements is displayed in Table S4.
Cis-acting elements involved in defense and stress responsiveness, such as drought-responsive elements (MBSs), anaerobic-responsive elements (AREs), low temperature-responsive elements (LTRs), and stress-responsive elements (TC-rich repeats), were detected in the promoters of the BchSOD genes. Furthermore, the drought-responsive element MBS was found primarily in the promoters of the BchMSD group and BchFSD genes. Moreover, many phytohormone-related cis-acting elements, which included abscisic-acid responsiveness (ABRE), MeJA-responsiveness (TGACG motif and CGTCA motif), auxin responsiveness (TGA element), gibberellin responsiveness (GARE motif and P-box) and salicylic-acid responsiveness (TCA element), were identified in the promoter region of the BchSOD genes. Most of the BchSOD genes were identified by analysis as having the MeJA-responsive TGACG motif (except for BchCSD1 and BchCSD4) and the CGTCA motif (except for BchCSD1, BchCSD4, and BchFSD5). Elements related to the auxin response are dispersed only within the promoters of MSD and CSD subfamily genes. Elements related to abscisic acid, gibberellin, and salicylic-acid responses are widely distributed among most BchSOD genes. These findings suggest that auxin plays a supervisory role in the MSD and CSD subfamilies and that phytohormones may have a regulatory effect on the control of BchSOD gene expression. Moreover, seven light-responsive genes (G-box, GT1-motif, Box 4, I-box, AE-box, chs-CAM1a, and TCT-motif) were found in the BchSOD gene promoter, among which I-box, AE-box, chs-CAM1a, and TCT-motif elements were distributed only in the BchCSD subfamily and BchFSD subfamily genes. Overall, the study showed that BchSOD genes are involved in the regulation of gene expression at different developmental stages and in the response to light, phytohormones, and abiotic stress in pakchoi.

3.5. Chromosomal Locations and Synteny Analysis of BchSOD Genes

To understand the tandem and segmental relationships of SOD genes in pakchoi, we further analyzed the chromosomal locations of BchSODs. Therefore, the chromosomal locations of 13 BchSOD gene pairs are shown in Figure 4. Eight out of the ten chromosomes harbored BchSODs. Detailed analysis revealed that chromosomes A03, A04, A05, A07, and A10 each contained one gene, chromosomes A01 and A09 each contained two genes, and chromosome A09 contained four genes. Moreover, one tandem repeat gene cluster in the BchSOD family was located on the A09 chromosome (BchFSD4 and BchFSD5). With the exception of BchMSD1 and BchCSD5, the remaining 11 genes had fragment duplication pairs (BchMSD2/BchMSD3, BchCSD3/BchCSD4, BchCSD1/BchCSD2, BchFSD1/BchFSD2, and BchFSD3/BchFSD5). This finding illustrates the importance of fragment duplication events in BchSOD diversity.
A collinearity analysis revealed many orthologs of SOD genes among B. chinensis and two closely related species (A. thaliana and B. oleracea) (Figure 5). The analysis revealed that nine B.chinensis genes exhibited synteny with AtSODs and BolSODs. Moreover, the BchSOD gene family had 17 and 11 homologous gene pairs with those of the BolSOD and AtSOD genes, respectively. In conclusion, numerous AtSOD and BolSOD homologs exhibited syngeneic relationships with BchSODs, demonstrating that genome-wide duplications or segmentation play essential roles in the evolution of the BchSOD gene family.

3.6. Analysis of the 2D and 3D Structures of BchSOD

Protein sequence structure can reflect protein function and lay the foundation for protein tertiary structure models. The 2D and 3D structures of the BchSOD proteins were analyzed (Figure S1 and Table S5), and the BchSOD protein structures were all composed of alpha helices, extended strands, beta turns, and random coils. Among these structures, those located in the same subfamily had similar structural models, while those in different subfamilies exhibited significant differences in structure. The predicted Mn-SOD subfamily (BchMSD1-BchMSD3) protein models and Fe-SOD subfamily (BchFSD1-BchFSD5) protein models are mainly composed of alpha helices and contain a small amount of beta-turns. In contrast, the Cu/Zn-SOD (BchCSD1-BchCSD5) model is composed mainly of highly conserved beta-turns and contains some short alpha-helices.

3.7. Expression Patterns of BchSOD Genes in Different Tissues

The tissue-specific expression of the BchSOD genes was assessed at four different tissue sites (comprising the roots, petioles, cotyledons, and true leaves) of pakchoi by qRT-PCR. As illustrated in Figure 6 and Table S6, we have found that the CSD and MSD subgroups, except for BchCSD5, were expressed at low levels in petioles and cotyledons. Compared to those of other genes, the expression levels of BchCSD3 and BchCSD4 were lower in true leaves. Moreover, a high number of FSD subgroup genes were expressed in all four parts of the test, and the expression was greater in the true leaves than in the other parts. Additionally, BchFSD1 was highly expressed in petioles, while other BchSOD genes exhibited decreased expression. This finding suggested that the FSD subgroup may have a vital effect on the growth of pakchoi.

3.8. Expression of BchSODs in Response to Abiotic Stresses and Exogenous Phytohormones

To explore the expression levels of BchSOD genes in response to different phytohormone (MeJA and GA) and abiotic stress (salinity and drought) treatments, 13 BchSOD genes were analyzed via qRT-PCR to determine the transcriptional profiles (Figure 7 and Table S7). BchSODs exhibited diverse expression patterns under various stress conditions. Under salt stress, there was a significant upregulation of BchCSD3 expression, which reached a maximum expression level at 12 h after Nacl treatment that was 3.97 times greater than that of the control. BchCSD2 and BchFSD2 were significantly upregulated at 48 h but not at other time points. Moreover, the expression of all the genes in the MSD subgroup did not change significantly. Under drought conditions, BchCSD3, BchCSD4, and BchCSD5 exhibited similar expression trends, with all exhibiting high expression at 12, 24, and 36 h. BchFSD2 showed significant upregulation at 24 h but not at other time points. Moreover, BchFSD4 and BchFSD5 were significantly upregulated at 24 and 48 h, respectively. Under GA treatment, BchFSD1 expression was markedly upregulated at all treatment time points, and it reached a maximum expression level at 8 h after Nacl treatment that was 2.13 times greater than that of the control. Moreover, the expression of four BchSOD genes, BchCSD1, BchCSD2, BchCSD4, and BchFSD2, was significantly upregulated at 2 and 8 h. BchFSD4 and BchFSD5 were significantly upregulated at 2 h, but not at other time points, resulting in significant variations. Under MeJA treatment, nearly all CSD subgroup and FSD subgroup member genes, except BchCSD1, BchCSD2, and BchFSD1, had relatively high expression levels. These results suggest that most BchSOD genes, especially those in the CSD and FSD subgroups, are involved in the phytohormone and abiotic stress response.

3.9. Subcellular Localization of the BchFSD2 Protein

Through preliminary analysis, we selected BchFSD2, which is abundantly expressed in leaves and significantly associated with abiotic stresses such as salt and drought, as well as hormones such as GA and MeJA, for subcellular localization studies. The cellular distribution of the BchFSD2-GFP fusion protein in the tobacco epidermis was further explored using the Agrobacterium-mediated tobacco transient expression system. The GFP signature of the control expression vector was present in the nucleus, plasma membrane, and chloroplasts at the same time, and the BchFSD2-GFP fusion protein was expressed only in chloroplasts after transient expression in tobacco (Figure 8). This result suggested that the BchFSD2 gene functions in chloroplasts.

3.10. Overexpression of the BchFSD2 Gene Improves Salt Stress Tolerance in Arabidopsis thaliana

To further investigate the function of the BchFSD2 gene under salt stress, we constructed a BchFSD2 overexpression vector to transform Arabidopsis thaliana and investigated the gene function through changes in the phenotypes and physiological indices of the transgenic plants under salt stress (Figure 9). We subjected the transgenic plants (L1) and the WT plants to salt stress and established a control group for both L1 and WT, and significant phenotypic differences were observed between the plants after 9 days of salt stress treatment (Figure 9A). After PCR verification, it was determined that the target gene was successfully transferred into Arabidopsis thaliana (Figure 9B). Compared with those of the control plants, both the WT and L1 salt-stressed plants presented a yellow leaf color and plant dwarfing phenotype, but L1 presented fewer symptoms and was less inhibited than WT.
Furthermore, when plants are subjected to abiotic stresses such as salinity, massive quantities of ROS can accumulate in the plant body and threaten their growth, while the plant itself will maintain its own order through the production of antioxidants; thus, ROS and antioxidants are important indicators of the degree of injury to plants. Therefore, we measured CAT, POD, and SOD activities as well as superoxide anion and MDA contents (Figure 9C–G). During salt stress, the superoxide anion and MDA contents in L1 were significantly lower than those in WT, while in contrast, the antioxidant activity of CAT, POD, and SOD in L1 was markedly greater than that in WT. In conclusion, the differences in phenotypic and physiological indices suggest that the increase in salt tolerance in BchFSD2 overexpressing plants may be mainly realized through the reduction of oxidative damage.

4. Discussion

Pakchoi is an important crop that is widely grown and consumed in Asia because it is highly nutritious and can be consumed in a variety of ways [23]. Problems such as drought, high temperature, and salt stress can cause ROS accumulation in plants, which seriously affects the growth and development of pakchoi. In plant cells, SOD is a key enzyme in the first line of defense against ROS and is vital to plant physiological and biochemical processes because it plays a central role in protecting against the toxic effects of ROS generated by various environmental stresses [24]. Previous studies have all shown that SOD gene overexpression enhances resistance to abiotic stresses in plants such as Arabidopsis [25] and tobacco [26]. Current research on the SOD gene family in pakchoi is limited, and a complete exploration of SOD gene family characteristics and functions in pakchoi would be beneficial for enhancing the understanding of the SOD gene family in plants. Moreover, identifying and screening the best candidate genes associated with the response to abiotic stresses, such as salt stress in pakchoi, and performing in-depth analysis will be beneficial for subsequent investigations into how pakchoi responds to abiotic stresses.
In this study, we identified 13 SOD genes from the pakchoi genome, including three Mn-SOD genes, five Cu/Zn-SOD genes, and five Fe-SOD genes (Table 1), which were categorized into three groups according to their structural domains, namely MSD, CSD, and FSD (Figure 1). Consistent with previous findings, the number of SOD family gene members in angiosperms has not expanded substantially during evolution and shows a highly conserved state; for example, eight SOD genes in Arabidopsis [27], thirteen SOD genes in maize [28], and nine SOD genes in tomato [11]. There may be some differences in the number of SOD genes in different plants due to the timing of gene duplications, including tandem and segmental duplications, as well as the effects of species evolution. The discovery of SOD gene doubling in a variety of plants by other researchers [10,29], in conjunction with our analyses, suggests that gene duplication may play a vital role in the evolution of SOD genes.
The three subfamilies of the BchSOD family also exhibited a high degree of conservation in terms of gene structure. By analyzing the number and distribution of exons and introns in the BchSOD genes, several differences in the number of exons and introns were found, but the differences within the same subfamily were small. Previous studies have shown that changes in exons and introns are the driving force of gene evolution and also directly lead to structural differences [30]. Further analysis also revealed that the intron-exon distribution patterns were similar within the same subfamily of the BchSOD family (Figure 2C). All these findings indicate that BchSOD was conserved during the evolutionary process. In addition, the analysis of the conserved structural domains showed that certain motifs existed only in certain subfamilies; for example, motif 9 was predicted only in the Mn-SOD subfamily, but some of the same motifs (motifs 1, 3, and 4) were also found in the Fe-SOD and Mn-SOD subfamilies (Figure 2B). These results suggest that BchCSD and the BchFSD/BchMSD may have different origins. In addition, motif analysis revealed that motifs 6, 7, and 9 were more conserved, and further analysis showed that members of the Fe-SOD and Mn-SOD subfamilies were more conserved (Figure 2D).
From the perspective of species evolution, gene duplication occurs in different ways throughout the evolutionary process and is an important pathway that drives the evolution of species [31]. The 13 BchSOD genes were analyzed at chromosomal locations, and all 11 genes had segmental duplication pairs except BchMSD1 and BchCSD5 (Figure 4). Additionally, by studying the collinearity of Pakchoi with closely related species (A. thaliana and B. oleracea), many BchSOD gene families were shown to have homologous gene pairs with BolSOD and AtSOD (Figure 5). These findings suggest that both segmental duplications and genome-wide duplications play important roles in the developmental evolution of the BchSOD family.
BchSODs are functionally diverse, and proteins play crucial roles in determining molecular function, both in terms of structure and properties [31]. Physical characterization of the BchSOD proteins revealed significant differences in protein sequence length, molecular weight, instability index, and isoelectric point among the 13 BchSOD proteins (Table 1). We also predicted and analyzed the 2D and 3D structures of the BchSOD proteins to gain a deeper understanding of the BchSOD proteins. According to our protein structure prediction (Figure S1 and Table S5), beta turns accounted for a greater percentage of the structure in the Cu/ZnSOD models than in the other two subfamilies, and their corresponding ligands were present in the structural models of the different subfamily proteins, with the exception of BchFSD1, BchFSD4, and BchFSD5. These predictions are consistent with previous 3D modeling of SODs in rapeseed [10], rice [32], and water lily [33].
To better understand the regulation of SOD gene expression in Pakchoi, we analyzed the cis-acting elements in the promoter. A large number of light-responsive, defense and stress, and hormone-responsive elements were found in the promoter region of the BchSOD gene through the prediction of 13 BchSOD gene promoter regions (Figure 3 and Table S4). The presence of drought-responsive elements (MBSs), anaerobic-responsive elements (AREs), low temperature-responsive elements (LTRs), and stress-responsive elements (TC-rich repeats) among the defense and stress-responsive related elements is in agreement with the fact that plant SOD genes play vital roles in plant defense and stress response [24]. A large number of researchers have shown that in plants such as Arabidopsis thaliana [34], rice [35], and tobacco [36], SOD genes contribute to plant resistance to abiotic stresses. In addition, the promoter regions of the BchSOD genes were found to contain a series of cis-acting elements associated with phytohormone responses, such as abscisic- acid responsiveness elements (ABREs), MeJA- responsiveness elements (TGACG motif and CGTCA motif) and auxin-responsiveness elements (TGA elements). Previous researchers have also determined that SOD in plants is regulated by hormones such as ABA and MeJA by studying different plants [37,38,39,40]. It has also been found that gene expression in SOD transgenic tobacco plants is regulated by light [41], a phenomenon that is also in agreement with the abundance of light-related cis-acting elements we found in the BchSOD promoters. In conclusion, by analyzing cis-action elements, BchSODs were shown to be involved in the regulation of gene expression at different developmental stages in Pakchoi and to play an important role in the process of stress tolerance. These findings facilitate our understanding of BchSODs under different environmental conditions.
Plant SOD gene expression levels significantly change in different tissues and under different environmental conditions [10,11,34,42]. Therefore, we utilized qPCR to assess the tissue-specific expression levels of the BchSOD genes in four different regions (roots, petioles, cotyledons, and true leaves) (Figure 6 and Table S6). Compared with those of the other subfamilies, the FSD subfamily genes exhibited greater expression at all four sites, especially at the true leaf site. This result is comparable to the previous expression of tobacco SOD genes in different tissues [7]. In conclusion, the spatiotemporal expression of BchSOD family genes suggested that these genes are involved in different physiological activities.
Similarly, significant differences in BchSOD gene expression were detected under different hormones (GA and MeJA) and abiotic stress conditions (salt stress and drought stress) (Figure 7 and Table S7). Under salt stress and drought conditions, almost all the BchSOD genes, except for the BchMSD2 gene, were upregulated within 48 h after treatment, especially BchCSD3, BchCSD4, and BchFSD2. These results are in accordance with the results of previous studies. SOD genes are upregulated in both tomato and wheat plants under drought and salt stress conditions [11,43]. Different SOD genes were also found in maize at different times in response to salt and drought stress, suggesting that ZmSODs may have different regulatory mechanisms in response to salt and drought stress [28]. Taken together, the results of previous studies and our findings demonstrate that SOD genes exert conserved effects on plant abiotic stress and that different SOD genes have different mechanisms involved in the response to stress. Additionally, in the hormonal (GA and MeJA) environments, all the genes were upregulated from 0 h to 8 h, except for BchMSD3 and BchCSD3, which were downregulated GA treatment. Moreover, we found that the expression of several genes (BchCSD1, BchCSD2, BchCSD4, and BchFSD1) significantly increased in response to GA treatment, while the expression of the BchCSD3, BchCSD4, BchFSD2, BchFSD3, BchFSD4, and BchFSD5 genes significantly increased in response to externally applied MejA. Several studies have already shown that phytohormones can resist stress by activating signaling transcriptional pathways [44]. Similarly, in tea trees, several CSD and FSD genes were found to respond to exogenous GA and MejA, and the majority of SOD genes were upregulated in rapeseed in response to GA, ABA, and other stress treatments [10,45]. According to previous studies and analyses, most of the BchSOD gene expression is regulated by external hormones and abiotic stresses, and the stress resistance of Pakchoi plants is enhanced by increased gene expression.
Based on previous studies on the BchSOD gene family, we found that BchFSD2 was not only abundantly expressed on leaves but also sensitive to salt stresses; therefore, we hypothesized that the BchFSD2 gene may play an essential role in the response to abiotic stress in pakchoi. Phenotypic and physiological parameters of control and BchFSD2 gene overexpressing Arabidopsis plants subjected to simulated salt stress indicated that the BchFSD2 gene indeed plays a role in plant resistance to salt stress and that it likely enhances plant resistance to salt stress and that it likely enhances plant tolerance to salt stress by increasing the ROS removal activity of the plant (Figure 9 and Figure 10). The overexpression of SOD genes in plants such as tobacco [46,47,48], Arabidopsis thaliana, and tall fescue has been previously performed, and the transgenic plants were found to exhibit similar characteristics increase plant oxidative stress protection by enhancing the reactive oxygen species scavenging system, which is consistent with our findings.

5. Conclusions

In this study, a comprehensive gene family-wide analysis of the SOD gene family of pakchoi was performed, and 13 BchSOD genes were identified. The physicochemical properties, phylogeny, gene structure, conserved structural domains, cis-elements, chromosomal localization and collinearity relationships, tissue-specific expression, and expression profiles of the BchSOD genes were investigated, and it was concluded that the BchSOD genes play essential roles in the response to hormonal and abiotic stresses. Moreover, the BchFSD2 gene, which is sensitive to salt, will be studied in depth as a key candidate gene for plant resistance to abiotic stresses. The overexpression of the BchFSD2 gene in Arabidopsis thaliana increased the plant antioxidant enzyme activity, thereby increasing plant tolerance to salt stress. These findings provide new possibilities for BchFSD2 to regulate salt tolerance in pakchoi and provide a new theoretical basis for a more comprehensive understanding of SOD gene function in pakchoi.

Supplementary Materials

The following supporting information can be downloaded at https://www.mdpi.com/article/10.3390/agronomy14020384/s1, Table S1: A list of primers used for gene expression analysis by qRT-PCR. Table S2: A list of primers used for gene-specific amplification. Table S3: Detailed information on 10 identified motifs in BchSOD proteins. Table S4: Information on hormone- and stress-related cis-elements detected in the promoter regions of BchSOD genes. Table S5: Detailed information on the 2D and 3D structures of BchSOD proteins. Table S6: The expression profiles of BchSOD genes in different tissues. Table S7: The expression profiles of BchSOD genes under abiotic stresses and exogenous phytohormones. Figure S1. Predicted 3D structures of the BchSOD proteins. The dots in the figure represent ligands, the blue bars represent alpha helices, and the green bars represent beta turns. Figure S2. Phylogenetic tree of SOD proteins from A. thaliana, B. oleracea, B. napus, and B. chinensis. (the maximal method). Figure S3. Phylogenetic tree of SOD proteins from A. thaliana, B. oleracea, B. napus, and B. chinensis. (the minimal method).

Author Contributions

Conceptualization, F.Z., Y.Z., M.H. (Maomao Hou) and S.L.; methodology, Y.Z.; software, S.R. and Y.Z.; validation, Y.X. and Y.Z.; formal analysis, Y.Z.; investigation, M.H. (Mingxuan Han); resources, S.L.; data curation, S.R.; writing—original draft preparation, Y.Z.; writing—review and editing, S.L., Y.Z. and F.Z.; visualization, Y.Z.; supervision, S.R.; funding acquisition, F.Z. All authors have read and agreed to the published version of the manuscript.

Funding

This work was supported by the National Key Programs for Bok Choy Breeding of China (111821301354052283), the Fujian Modern Agricultural Vegetable Industry System Construction Project (2019-897), the Seed Industry Innovation and Industrialization Project in Fujian Province (zycxny2021009), and the Fuzhou Science and Technology Project (2022-P-001; 2022-P-018).

Data Availability Statement

Data are contained within the article and Supplementary Materials.

Conflicts of Interest

The authors declare no conflicts of interest.

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Figure 1. Phylogenetic tree of SOD proteins from A. thaliana, B. oleracea, B. napus, and B. chinensis (the neighbor-joining (NJ) method). Different colors represent different branches: Fe-SOD (blue), Cu/Zn-SOD (red), and Mn-SOD (yellow). Moreover, BchSOD is labeled with a red star. At: Arabidopsis thaliana, Bol: Brassica oleracea, Bn: Brassica napus, Bch: Brassica chinensis.
Figure 1. Phylogenetic tree of SOD proteins from A. thaliana, B. oleracea, B. napus, and B. chinensis (the neighbor-joining (NJ) method). Different colors represent different branches: Fe-SOD (blue), Cu/Zn-SOD (red), and Mn-SOD (yellow). Moreover, BchSOD is labeled with a red star. At: Arabidopsis thaliana, Bol: Brassica oleracea, Bn: Brassica napus, Bch: Brassica chinensis.
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Figure 2. Phylogenetic tree, conserved motifs, gene structures, and motif logos of BchSODs. (A) Phylogenetic tree of BchSODs. (B) Conserved motifs of BchSODs. Ten motifs in BchSOD were detected using the MEME tool and were labeled with different colors. (C) Gene structure of BchSODs. The gray lines represent introns, and the green and gray boxes represent CDSs and UTRs, respectively. (D) Logos of ten detected motifs of BchSODs.
Figure 2. Phylogenetic tree, conserved motifs, gene structures, and motif logos of BchSODs. (A) Phylogenetic tree of BchSODs. (B) Conserved motifs of BchSODs. Ten motifs in BchSOD were detected using the MEME tool and were labeled with different colors. (C) Gene structure of BchSODs. The gray lines represent introns, and the green and gray boxes represent CDSs and UTRs, respectively. (D) Logos of ten detected motifs of BchSODs.
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Figure 3. Cis-acting in the promoters of BchSOD genes. (A) Different color cubes display different cis-acting related to various defense and stress responsiveness, light responsiveness, and phytohormone responsiveness. (B) The frequencies of cis-acting elements are indicated by numbers and different colors.
Figure 3. Cis-acting in the promoters of BchSOD genes. (A) Different color cubes display different cis-acting related to various defense and stress responsiveness, light responsiveness, and phytohormone responsiveness. (B) The frequencies of cis-acting elements are indicated by numbers and different colors.
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Figure 4. Chromosomal locations and interchromosomal associations of BchSODs. The Circus diagram is from the inside out, with the first and second layers being chromosome density and the third layer being the chromosomes. The gray background inside the circle represents all syntenic blocks in the B. chinensis genome, and the red lines represent homozygous BchSOD gene pairs.
Figure 4. Chromosomal locations and interchromosomal associations of BchSODs. The Circus diagram is from the inside out, with the first and second layers being chromosome density and the third layer being the chromosomes. The gray background inside the circle represents all syntenic blocks in the B. chinensis genome, and the red lines represent homozygous BchSOD gene pairs.
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Figure 5. Collinearity analysis of SOD genes in A. thaliana, B. chinensis, and B. oleracea chromosomes. The grey areas in the figure indicate the collinear regions in B. chinensis and other plant genomes, and the red lines display the syntenic SOD gene pairs.
Figure 5. Collinearity analysis of SOD genes in A. thaliana, B. chinensis, and B. oleracea chromosomes. The grey areas in the figure indicate the collinear regions in B. chinensis and other plant genomes, and the red lines display the syntenic SOD gene pairs.
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Figure 6. Relative expression levels of BchSOD genes in different pakchoi tissues. Red represents a high expression level, and blue represents a low expression level.
Figure 6. Relative expression levels of BchSOD genes in different pakchoi tissues. Red represents a high expression level, and blue represents a low expression level.
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Figure 7. Expression of BchSOD genes under different abiotic stress and phytohormone conditions at different time points. The abiotic condition (salinity and drought) time points were 0 (CK), 12, 24, 36, and 48 h, and the phytohormone control (GA and MeJA) time points were 0 (CK), 2, 4, 6, and 8 h. The expression bands show comparative gene expression tendencies based on log2 fold change values. Red represents high expression, and blue represents low expression.
Figure 7. Expression of BchSOD genes under different abiotic stress and phytohormone conditions at different time points. The abiotic condition (salinity and drought) time points were 0 (CK), 12, 24, 36, and 48 h, and the phytohormone control (GA and MeJA) time points were 0 (CK), 2, 4, 6, and 8 h. The expression bands show comparative gene expression tendencies based on log2 fold change values. Red represents high expression, and blue represents low expression.
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Figure 8. Subcellular localization of BchFSD2 in tobacco epidermal cells. BchFSD2-GFP and 35s-GFP represent confocal images of the control empty vector and transient BchFSD2 expression, respectively. GFP is the fluorescence field, Chloroplast is the chloroplast field, Bright is the bright field, and Merge is the superimposed field. The scale bar represents 50 μm.
Figure 8. Subcellular localization of BchFSD2 in tobacco epidermal cells. BchFSD2-GFP and 35s-GFP represent confocal images of the control empty vector and transient BchFSD2 expression, respectively. GFP is the fluorescence field, Chloroplast is the chloroplast field, Bright is the bright field, and Merge is the superimposed field. The scale bar represents 50 μm.
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Figure 9. Overexpression of BchFSD2 in Arabidopsis thaliana. (A) Plant growth phenotypes under salt stress. WT: wild type subjected to salt stress, L1: transgenic phenotype subjected to salt stress, WT-CK: wild type subjected to normal watering, L1-CK: normal watering transgenic type. (B) PCR validation plot of the overexpressed BchFSD2 gene in transgenic plants. (C) CAT activity. (D) POD activity. (E) SOD activity. (F) Superoxide anion content. (G) MDA content. The data are presented as the standard errors of the means of three independent experiments (n = 3). Different letters indicate significant differences between treatments at p < 0.005.
Figure 9. Overexpression of BchFSD2 in Arabidopsis thaliana. (A) Plant growth phenotypes under salt stress. WT: wild type subjected to salt stress, L1: transgenic phenotype subjected to salt stress, WT-CK: wild type subjected to normal watering, L1-CK: normal watering transgenic type. (B) PCR validation plot of the overexpressed BchFSD2 gene in transgenic plants. (C) CAT activity. (D) POD activity. (E) SOD activity. (F) Superoxide anion content. (G) MDA content. The data are presented as the standard errors of the means of three independent experiments (n = 3). Different letters indicate significant differences between treatments at p < 0.005.
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Figure 10. BchFSD2 gene overexpression regulating the modeling diagram of pakchoi tolerance to salt stress. The blue dashed box represents the changes in plants under salt stress.
Figure 10. BchFSD2 gene overexpression regulating the modeling diagram of pakchoi tolerance to salt stress. The blue dashed box represents the changes in plants under salt stress.
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Table 1. The characteristics of 13 SOD genes in pakchoi.
Table 1. The characteristics of 13 SOD genes in pakchoi.
Gene
Name		Gene IDGenomic Position (bp)Protein
Length (aa)		CDS
Length (bp)		Molecular
Weight/(Da)		Isoelectric
Point (PI)		GRAVYInstability
Index		Subcellular
Localizations
BchMSD1Bch09G054140.1A09-53671833:53673122(−)24172660.265.110.85155.34Extracellular
BchMSD2Bch01G046450.1A01-34214509:34215993(−)23169656.595.140.76641.68Mitochondrial
BchMSD3Bch05G042360.1A05-35515870:35517513(−)23169656.765.150.73644.12Mitochondrial
BchCSD1Bch06G006380.1A06-3784500:3786799(+)15345937.835.210.73457.62Cytoplasm
BchCSD2Bch09G070780.1A09-61463615:61465502(−)15245937.955.210.70453.29Cytoplasm
BchCSD3Bch07G020850.1A07-18191165:18192695(+)16248942.125.141.01766.86Chloroplast
BchCSD4Bch04G023970.1A04-18398587:18400291(+)20762452.885.110.86458.93Chloroplast
BchCSD5Bch10G024890.1A10-20006809:20008560(−)16248940.155.210.76257.07Cytoplasm
BchFSD1Bch03G016650.1A03-7871955:7873744(+)29990073.45.10.80247.6Chloroplast
BchFSD2Bch01G016870.1A01-8554165:8556874(+)26479566.655.080.89761.46Mitochondrial
BchFSD3Bch06G036350.1A06-38063266:38064684(+)26379265.085.140.71349.96Chloroplast
BchFSD4Bch09G007260.1A09-3927405:3929188(−)26379265.25.140.70851.3Chloroplast
BchFSD5Bch09G007220.1A09-3910081:3911975(−)26981066.765.140.68749.65Chloroplast
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Zhou, Y.; Li, S.; Ran, S.; Xu, Y.; Hou, M.; Han, M.; Zhong, F. Genome-Wide Identification and Characterization of the Superoxide Dismutase (SOD) Gene Family in Pakchoi and the Role of the BchFSD2 Gene in the Salt Stress Toleran. Agronomy 2024, 14, 384. https://doi.org/10.3390/agronomy14020384

AMA Style

Zhou Y, Li S, Ran S, Xu Y, Hou M, Han M, Zhong F. Genome-Wide Identification and Characterization of the Superoxide Dismutase (SOD) Gene Family in Pakchoi and the Role of the BchFSD2 Gene in the Salt Stress Toleran. Agronomy. 2024; 14(2):384. https://doi.org/10.3390/agronomy14020384

Chicago/Turabian Style

Zhou, Yuqi, Shuhao Li, Shengxiang Ran, Yang Xu, Maomao Hou, Mingxuan Han, and Fenglin Zhong. 2024. "Genome-Wide Identification and Characterization of the Superoxide Dismutase (SOD) Gene Family in Pakchoi and the Role of the BchFSD2 Gene in the Salt Stress Toleran" Agronomy 14, no. 2: 384. https://doi.org/10.3390/agronomy14020384

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